Olagunju et al.

Food Production, Processing

Food Production, Processing and Nutrition (2024) 6:32
https://doi.org/10.1186/s43014-023-00175-8 and Nutrition

RESEARCH Open Access

High‑protein, low glycemic index snack

from optimized blend of three wholegrains
exhibits nutraceutical quality and elicits low
glycemic response in diabetic human subjects
Aderonke Ibidunni Olagunju1* , Titilope Ifeolu Arigbede1, Idowu Sunday Oyeleye2,
Solomon Akinremi Makanjuola3, Esther Taiwo Oyebode1 and Adenike Christianah Enikuomehin4

Abstract
Snack products are evolving as new carriers of functional ingredients with nutritional and health-promoting benefits.
A blend of whole grains is increasingly being utilized to harness the functional potential of the grain mix. Amaranth,
acha, and pearl millet grains flours were optimized using response surface methodology (RSM), to obtain opti-
mum blends (90:5:5 and 47.98:26.68:25.34) with high protein content and low glycemic index. Snack bar products
from the blends were labelled MBY and MBZ. A total of 40 diabetic and 10 non-diabetic subjects were recruited.
Of the diabetic, about 42% were overweight while 40% were obese, the non-diabetic had normal weights. Each
was allowed to consume snacks containing the equivalent of 50 g of carbohydrates. Finger prick was employed
to evaluate the postprandial glucose response of snack products while venous blood was evaluated for antioxidant
enzymes, carbohydrate-hydrolyzing activities, and insulin using standard methods. Consumption of the multigrain
snacks elicited a stable postprandial response (133–141 mg/dL) with 16 and 24% postprandial decline. In addition,
snacks had low to intermediate glycemic index (52 and 56) in diabetic and low glycemic index (43 and 45) in non-
diabetics; likewise reduced α-amylase/α-glucosidase activities compared to control snacks. Similarly, glutathione
level, glutathione peroxidase, superoxide dismutase, and catalase activities in serum from subjects that consumed
multigrain snacks were upregulated compared to control and market sample groups. Moreso, snack products pro-
moted a reduction in serum insulin levels in diabetic subjects (45 and 17% for MBY and MBZ respectively). Following
the nutraceutical properties displayed by the formulated snack especially MBY, it can be promoted as a functional
snack for the management of diabetes while solving the limited snack product choice of diabetes sufferers.
Keywords Multigrain, High-protein snack, Clinical observation, Glycemic regulation, Serum biochemical properties

*Correspondence:
Aderonke Ibidunni Olagunju
aiolagunju@futa.edu.ng
Full list of author information is available at the end of the article

© The Author(s) 2024. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which
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Olagunju et al. Food Production, Processing and Nutrition (2024) 6:32 Page 2 of 17

Graphical Abstract

Introduction be over-emphasized. Several reports have shown the

Diabetes mellitus (DM), which is regarded as the dis- effectiveness of a high-protein plant regimen in reduc-
ease of the 2 ­ 1st Century, is a growing epidemic health ing the prevalence of diabetes in human subjects, some
problem, characterized by elevated blood glucose as a of which are Malaeb et al. (2019) and Yang et al. (2021).
result of either insufficient insulin production by the In a similar fashion, low glycemic index foods are report-
pancreas or a result of ineffective use of the insulin pro- edly beneficial for blood glucose control (Ojo et al. 2018;
duced (WHO 2021). Changes in global food systems, Bai et al. 2021). Plant-based diets include products from
the increasing adoption of high caloric intake and low legumes, whole grains, vegetables and fruits, are nutri-
energy expenditure (sedentary lifestyle) have promoted tious and economically sustainable with diverse product
the pervasiveness of obesity and type-2 DM (Hruby & options. Whole grain comprising the bran and germ frac-
Hu 2015). Improper management of DM has resulted in tions possess outstanding nutritional and bioactive prop-
several macrovascular and microvascular blood vessels erties, conferring relevant health-promoting benefits to
initiating nephropathy, ocular disorders, heart attack, its consumers and is relevant in weight control (Calinoiu
stroke and eventually death (Petrie et al. 2018). Obesity & Vodnar 2018).
and hyperlipidemia are key risk factors of DM, thus, Globally, snacks are an important contributor to
weight management is an essential tool for effective human’s daily energy intake (Smith & Whiting 2019).
diabetic care and its complications, including cardio- They are often regarded as junk foods but recently,
vascular diseases such as blood pressure, arteriosclero- snack products are evolving as new carriers of func-
sis, and glycemic control (Wing et al. 2011). tional ingredients with nutritional and health-promoting
‘High protein’ foods contain above 11 g/100 g protein benefits (Kim et al. 2020). Impressively, the potential of
(Ministry of Food and Drug Safety 2019). Interestingly, functional foods including functional snack products in
increased protein intake through high-protein foods controlling hyperglycemia or managing diabetes is being
complemented with reduced carbohydrate intake has explored (Krawecka et al. 2019; Yang et al. 2021). The
the potential to reduce plasma glucose levels of diabetic consumption of healthy snacks reportedly controls the
individuals (Gannon & Nuttall 2004). Diet as one of the rise in blood glucose levels while controlling the glyce-
lifestyle interventions for diabetes management cannot mic response of the subsequent meal (Imai et al. 2018;
Olagunju et al. Food Production, Processing and Nutrition (2024) 6:32 Page 3 of 17

Nitta et al. 2019). The diet approach to the management Materials and methods
and prevention of type 2 diabetes in some countries is Amaranth (Amaranthus caudatus) and pearl millet (Pen-
remarkable (Hwalla et al. 2021; Rajput et al. 2022). The nisetum glaucum) were sourced from Shasha market,
glycemic index (GI) measures the dynamics of hydroly- Akure, Ondo State. Acha (Digitaria exilis) was sourced
sis and absorption of carbohydrates into the blood- from a local market in Minna, Niger State. The grains
stream whereas, glycemic load (GL) predicts the effect were authenticated by a Crop Scientist in the Depart-
of amount of carbohydrate consumed per serving ment of Crop, Soil, and Pest Management, at the Federal
(Krawecka et al. 2019). Overall, both are rating systems University of Technology Akure, Nigeria. Date syrup was
to measure the promptness of foods to cause increase in purchased from a local spice store. Coconut oil, baking
blood glucose levels. Foods with low GI and GL promote powder and ginger powder were purchased from the
low glycemic response to carbohydrate-containing foods supermarket. Soy lecithin was sourced from Qualifirst
and are associated with reduced risk of diseases such Foods Ltd, Toronto, Canada. All reagents were of analyti-
as type 2 diabetes mellitus and cardiovascular diseases cal grade.
(Augustin et al. 2015).
Cereal grains are specifically employed for the pro- Processing of flour samples
duction of baked products. Multicereal represents a Amaranth, acha, and pearl millet grains were manu-
combination of grains and may confer functional char- ally sorted, thereafter, thoroughly washed and steeped
acteristics derived from the synergistic contributions with distilled water in individual sterile containers. The
of the individual cereal grain. This may be a result of grains were subjected to fermentation (37 °C/72 h). Note
the rich content of dietary fibre, presence of functional that the steep water was changed daily. It was thereafter
phytochemicals and the low glycemic index (Prasadi & drained, allowed to dry in a laboratory oven under con-
Joye 2020). Baked food products especially from white vective heat at 45 °C for 6 h, then milled to coarse par-
flour with the inclusion of white sugar has been reported ticle size (212 µm). Flour was packed in screw-capped
to possess relatively high glycemic indices (Olagunju containers and stored at refrigeration temperature until
2019). However, with a careful selection of functional required for further use.
cereals and use of natural sweeteners, resulting snacks
may provide potential health benefits to diabetic and Multigrain flour blend formulation
normoglycemic individuals. Indigenous wholegrains Mixture design of response surface methodology (RSM)
with health-promoting properties beyond nutrients was employed for the optimization of flour blends, and
were carefully selected for this study. The grains have 14 experimental runs were generated (Table 1). The pre-
been individually studied and their positive role in the fermented coarse flour samples were mixed using a range
management of diabetes is worthy of endorsement. of 5 to 90% for the flour blend using protein content and
Amaranth, acha and pearl millet have received popu- glycemic index (GI) as dependent variables. The first
lar attention for their antidiabetic properties (Kasozi round of optimization to maximize protein and minimize
et al. 2018; Adams & Yakubu 2020; Pei et al. 2022). This the GI yielded the blend 90:5:5 (amaranth:acha:pearl
property is attributed to the richness in fibre and pro- millet), while a second round of optimization to set the
tein as well as the presence of complex carbohydrates protein and GI between 16–18% and 40–45 respectively
and the low glycemic nature (Saleh et al. 2013; Geetha yielded a blend 47.98:26.68:25.34 (amaranth:acha:pearl
et al. 2020). Given the respective nutritional relevance millet). The optimized blends were employed in the pro-
and health benefits of the selected grains, developing duction of snack bars labeled MBY and MBZ respec-
characteristic snack product from an optimized cereal tively while commercial oat snack bars (COB) served as
blend is a step in the right direction to meeting consum- control.
ers’ preference for a functional healthy snack product.
Snacking is a part of the dietary treatment regimen Production of snack bar
of diabetic patients however, snacks for these group of The dry ingredients were weighed into a bowl. On the
people remain unavailable in Nigeria. Hence, the study side, 10 ml of clean water was dispensed into a glass
aimed to develop a healthy nutritious snack product from beaker, followed by the addition of 10 g each of date syrup
underutilized functional cereals. More importantly, eval- and coconut oil respectively. The glass beaker was heated
uate their postprandial blood glucose effect and glycemic in a water bath (70 °C) till the mixture was uniformly dis-
index as consumption of low glycemic index foods may solved. The wet ingredients were then added to the dry
reduce the incidence of serious diabetic complications by ingredients and then completely mixed to form a paste.
abating insulin resistance and promoting strict glycemic The dough was rolled to a 3 cm thickness and then trans-
control in type 2 diabetes mellitus patients. ferred to a baking tray (30 cm × 30 cm). The dough sheet
Olagunju et al. Food Production, Processing and Nutrition (2024) 6:32 Page 4 of 17

Table 1 Flour composition, protein content, and glycemic index Chemical analysis of snack product
of RSM generated runs Proximate compositions (total ash - method no.930.22,
Runs Amaranth Acha Pearl millet Protein GI crude fibre - method no. 950.37, crude fat - method
no. 950.36, crude protein - method no. 950.36) of the
1 33.333 33.333 33.333 14.29 ± 0.29 43.59 ± 1.09 snack products were determined as described in AOAC
a
2 90.00 5.00 5.00 22.65 ± 0.17 42.52 ± 0.58 (2012). Carbohydrate content was calculated by differ-
3 19.167 61.667 19.167 12.35 ± 0.04 47.06 ± 0.48 ence. The proximate composition of the snack product
4 19.167 19.167 61.667 10.55 ± 0.21 52.86 ± 1.11 was evaluated, results reported in an earlier study (Ola-
5 5.00 90.00 5.00 10.84 ± 0.12 47.78 ± 0.63 gunju et al. 2022). The carbohydrate content (g/100 g
6 5.00 90.00 5.00 10.71 ± 0.06 52.14 ± 1.80 wet weight basis) of each test food was used in estimat-
7 47.50 5.00 47.50 15.89 ± 0.04 43.64 ± 1.03 ing the amount equivalent to 50 g carbohydrate. The car-
8 90.00 5.00 5.00 21.13 ± 0.10 41.62 ± 2.24 bohydrate content of COB was the least (42.34 g/100 g)
9 5.00 5.00 90.00 12.31 ± 0.08 58.00 ± 2.16 while MBY and MBZ had 44.95 and 46.83 g/100 g car-
10 5.00 47.50 47.50 7.93 ± 0.04 38.64 ± 0.50 bohydrate respectively (Table 3 shows dry weight basis
11 47.50 47.50 5.00 16.24 ± 0.13 43.72 ± 0.76 composition).
a
12 47.98 26.68 25.34 16.95 ± 0.03 43.54 ± 1.06 The study was designed to evaluate the potential
13 5.00 5.00 90.00 12.65 ± 0.02 56.00 ± 2.24 of multigrain snack products as dietary intervention
14 61.667 19.167 19.167 14.14 ± 0.02 48.16 ± 0.36 and as snacks for diabetic subjects. The study was con-
a
Indicates the blends selected for production of snack product; GI: glycemic ducted at the Medical Out-Patient Department of the
index University of Medical Sciences Teaching Hospital (UNI-
MEDTHC), Akure, and the Federal University of Tech-
was partitioned into a length and width of 6 cm × 2 cm. nology, Akure, Nigeria. The clinical study was carried out
Baking was carried out in a laboratory oven set at 150 °C following guidelines for human studies with the approval
for 30 min and the bars separated from each other after of the Ondo State Health Research Ethics Committee
baking. The snack bars were cooled and sealed in a poly- (OSHREC), Ondo State (OSHREC 22/3/2021/316). We
propylene film; placed in an airtight container and stored hereby certify that the study was performed in accord-
at room temperature. Assuredly, the developed multigrain ance with the 1964 Helsinki Declaration and comparable
snack was prepared under strict hygienic conditions a ethical standards.
day before the evaluation while the commercial product
was purchased from Ceci supermarket, Akure. The ingre- Basic characteristics and anthropometric measurements
dients and nutritional compositions of the snack bar are The age, sex, occupation of the patients were docu-
provided in Tables 2 and 3. The ingredient for the oat mented. The diabetic history (year of diagnosis and
snack (market sample which served as a control) com- treatment commencement) was also documented. The
prised whole grain rolled oat, brown sugar syrup, honey, weight of each subject was measured using a digital clinic
sunflower lecithin, sodium bicarbonate, salt, and sun-
flower oil.

Table 3 Proximate composition (dry weight basis) of whole

grain snack bar
Table 2 Formulation of ingredients for snack bar
Constituents (g/100 g) Samples
Ingredients (g/100 g) MBY (g) MBZ (g)
MBY MBZ COB
Amaranth flour (%) 90 47.98
Ash 3.19 ± 0.00a 2.45 ± 0.41b 1.39 ± 0.01c
Acha flour (%) 5 26.68
Crude fiber 2.08 ± 0.01b 1.08 ± 0.01c 3.54 ± 0.34a
Pearl millet flour (%) 5 25.34
Fat 13.55 ± 0.05a 17.86 ± 0.10b 25.06 ± 0.08a
Date syrup 10 10
Protein 21.95 ± 0.66a 17.75 ± 0.19b 3.54 ± 0.34c
Soy lecithin 1 1
Carbohydrate 59.23 ± 0.70b 60.86 ± 0.55a 56.49 ± 0.41c
Ginger powder 2 2 a a
Moisture 6.96 ± 0.28 7.03 ± 0.11 4.06 ± 1.29b
Baking powder 1 1 c b
Energy value (kcal/100 g) 446.67 ± 0.62 475.02 ± 1.04 464.58 ± 1.52a
Coconut oil 10 10
Vanilla extract 0.5 0.5 Values represent Mean ± SD, values with different superscripts on the same row
are significantly different (P˂ 0.05)
Xanthan gum 3 3
MBY (snack comprising 90% amaranth, 5% acha, 5% pearl millet); MBZ (snack
MBY: 90% amaranth, 5% acha, 5% pearl millet; MBZ: 47.98% amaranth, 26.68% comprising 47.98% amaranth, 26.68% acha, 25.34% pearl millet), COB Oat snack
acha, 25.34% pearl millet bar control (control)
Olagunju et al. Food Production, Processing and Nutrition (2024) 6:32 Page 5 of 17

weighing scale. Height was measured using a portable Group 2 (G2): diabetic subjects fed with multigrain
calibrated clinic stadiometer (Seca, mLabs209, Germany). snack bar from blend 1 comprising 90% amaranth,
The body mass index (BMI) was calculated as weight (kg) 5% acha, 5% pearl millet (MBY)
divided by the square of height (­m2). The systolic blood Group 3 (G3): diabetic subjects fed with multigrain
pressure (SBP) and diastolic blood pressure (DBP) of par- snack bar from blend 2 comprising 47.98% amaranth,
ticipants were measured using a sphygmomanometer. 26.68% acha, 25.34% pearl millet (MBZ)
Group 4 (G4): diabetic subject fed with commercial
Experimental procedure oat snack bar (COB)
Forty (40) healthy, diabetic, non-smokers (male and female)
were recruited from the Medical Out-Patient Depart- The test food was consumed within 15 min along
ment of UNIMEDTHC Akure, Nigeria. Their body weights with 75 cL of drinking water. The subjects maintained
ranged from 45 to 90 kg (averaging 73.15 ± 12.50 kg) while a seating position throughout the duration (about 3 h)
their body mass index (BMI) ranged from 23 to 35 kg/m2 of the experiment; they were prohibited from eating
(with an average value of 28.36 ± 4.65 kg/m2). The partici- and drinking till the session ended. Although, light
pants were divided into 4 groups, each group comprising activities such as reading a book, newspaper or mobile
10 subjects. The inclusion criteria for participation were phone surfing were allowed. At the end of the study
adults with diagnosis of type 2 diabetes, being older than period, 5 mL of blood was drawn from the veins; the
40 years of age and with a BMI above 23 kg/m2. The exclu- blood was centrifuged (2,000 rpm for 10 min) and the
sion criteria include being on medications for any chronic resulting serum was stored for subsequent biochemical
disease conditions or those affecting glucose tolerance, gas- analysis.
trointestinal disorders, pregnancy, breastfeeding, intoler-
ances, or allergies to any ingredient in the snack product. Evaluation of oxidative stress marker in serum
The participants voluntarily gave their consent after being The level of lipid peroxides in the serum was evaluated as
informed about the study. They were properly briefed about described by Ohkawa et al. (1979) and reported as thio-
the assessment and requested to observe a 12 h overnight barbituric acid substance (TBARS) level. Exactly 100 μl of
fast while avoiding alcohol consumption or engagement in blood serum was added to 200 μl of 8.1% sodium dodecyl
rigorous physical exercise before the test day. The experi- sulphate (SDS), followed by the addition of 500 μl each of
ment was carried out for non-consecutive days. On the 0.8% thiobarbituric acid and acetic acid solution (2.5 M
other hand, ten (10) healthy, non-smoking, male and female HCl, pH 3.4) into the test tube. The reaction mixture was
subjects who have not been diagnosed with diabetes with 45 heated (100°C) for 1 hr, absorbance taken at 532 nm in a
to 70 kg body weights (average of 63.10 ± 8.45 kg) and 20 to spectrophotometer.
25 kg/m2 BMI (averaged 21.56 ± 1.32 kg/m2) was recruited
from among members of staff, Federal University of Tech-
nology, Akure. Evaluation of glycemic index and glycemic load
Upon arrival of the study subjects at the test centre, the of multigrain snack product
anthropometric parameters of weight and height were The GI of the snack products was evaluated following
measured following standard procedures and BMI was the World Health Organization guidelines FAO/WHO
calculated. The fasting blood glucose was also taken in a (1998). After consumption of the test meal, blood glucose
relaxed condition using a finger prick blood sample and concentrations were measured in capillary whole blood
measured using a Kiptrack blood glucose monitor (Med- obtained by finger prick and measured using Kiptrack
Net GmbH Borkstraβe 10, 48,163 Münster, Germany). blood glucose monitor (MedNet GmbH Borkstraβe 10,
On the first day, participants were given glucose solution 48,163 Münster, Germany) in the fasted state (time 0),
(50 g glucose dissolved in 250 mL clean drinking water) after 15 min of complete ingestion of the snack then at
and blood glucose readings were taken for 2 h, subse- 30 min interval for 2 h (30, 60, 90, 120 min). The incre-
quently, the test food (quantity equivalent to 50 g carbo- mental area under curve (iAUC) for test food and glu-
hydrate) was administered to the participants according cose was calculated using the trapezoidal rule of the area
to their group stated as follows: under the curve to determine the GI was calculated using
the formula below:
Normal control (NC): non-diabetic healthy subjects Incremental area under 2h blood glucose curve for food samples
Group 1 (G1): diabetic subjects not administered GI =
Incremental area under 2h blood glucose curve for glucose
X 100

with any test food (CTRL)

The glycemic load (GL) for each food sample was
determined as described by Salmeron et al. (1997). GL
Olagunju et al. Food Production, Processing and Nutrition (2024) 6:32 Page 6 of 17

was estimated from the carbohydrate content in a typical Statistical analysis

serving of the test meal and the GI of the test food using Optimization was carried out using Response surface
the equation below: analysis; Design Expert v. 7.0.0 (Stat-Ease, Inc., Minne-
apolis, MN, USA). The dataset generated was analysed
Net carbohydrate g xGI using SPSS 25.0 for Windows software (SPSS Inc., Chi-
GL =
100 cago, IL, USA), the means were separated using Duncan’s
New Multiple Range Test (DNMRT) and the analysis of
Net Carbohydrate = Total Carbohydrate available in a
variance (ANOVA) was performed. The incremental area
serving of the food sample
under the curve (iAUC) was calculated and biochemi-
cal activities analysed using GraphPad Prism program
Determination of serum antioxidant parameters
for Windows (version 8.0; GraphPad Software, Inc., San
Serum glutathione peroxidase (GPx) activity was evalu-
Diego, CA, USA).
ated following the method of Rotruck et al. (1973). To
the prepared buffer, serum, and glutathione were added,
Results and discussion
respectively, and the mixture was incubated at 37 °C for
Basic characteristics
10 min. The mixture was centrifuged after the addition
The basic characteristics and anthropometric data of
of 10% trichloroacetic acid. The supernatant and Ellman’s
the study group (diabetic and non-diabetic subjects) are
reagent with phosphate buffer were added together and
presented in Table 4. The non-diabetic subjects were
the absorbance was taken at 412 nm.
volunteered academic and non-academic staff members
The reduced glutathione (GSH) level in the blood
of the Federal University of Technology, Akure, Nigeria.
serum was determined at 412 nm using the method
They comprise an equal number of males and females
described by Ellman (1959). This involved 100 µL of
aged 39 to 52 years old (average age 44.70 ± 4.94 years).
serum treated with 50 μL of Ellman’s reagent (19.8 mg of
Data showed they had a healthy and normal body weight
5,5′dithiobisnitrobenzoic acid in 100 mL of 0.1% sodium
averaging 63.10 ± 8.45 kg and BMI 21 to 22.81 kg/m2
citrate) and 300 µL of phosphate buffer (pH 8.0).
(average of 21.56 ± 1.32 kg/m2). The diabetic subjects
The serum superoxide dismutase (SOD) activity was
were volunteered patients at the out-patients’ depart-
determined at 480 nm based on the inhibition of the
ment of the University of Medical Sciences Teaching
superoxide radical produced in the presence of adrenalin
Hospital, Akure, Nigeria. Going by the statistics of the
according to the method by Misra and Fridovich (1972).
diabetic participants, the female gender was more rep-
Catalase activity in serum was evaluated following the
resented than the male counterpart with a sex ratio
method described by Beers and Sizer (1952), 50 μl of
of 1.2, and their age range was between 46 to 80 years
serum was added to a reagent mixture comprising 500 μl
(averaged 65.87 ± 9.69 years), the most represented age
of 59 mM ­H2O2 and 950 μl of 50 mM phosphate buffer
group was 51–60 and 71–80 years. About 32% (13 per-
[pH 7.0] before the reaction began. For three minutes,
sons) were civil servants (government employees), 17%
the reaction was allowed to proceed at 25°C while the
(7 persons) were entrepreneurs while 50% (20 persons)
absorbance was monitored every 150 s (570 nm).
were retirees thus, about half of the respondents had
working functional status. From the information pro-
Determination of serum insulin level and carbohydrate
vided by the patients, the diagnostic duration of diabe-
(α‑amylase and α‑glucosidase) enzymes activity
tes was 5 years on average. The average weight was 73 kg
Serum’s insulin was quantified using an ELISA kit
and average body mass index (BMI) was 28 kg/m2. The
(Monobind Inc, USA) following the manufacturer’s
male had a weight range between 69–90 kg and a BMI
instructions. The absorbance was measured at 450 nm
of 27.48–31.88 kg/m2 while female weighed 45–90 kg
in a microplate reader (BIOBASE-EL10A). The serum
and BMI 23.48–36.60 kg/m2. According to the World
α-amylase activity was evaluated using 1% soluble
Health Organization (WHO), BMI cut-offs and their
starch solution prepared phosphate buffer as substrate.
corresponding values are underweight (BMI < 18.5 kg/
After incubating the mixture with dinitrosalicylic acid
m2), normal weight (BMI = 18.5–24.9 kg/m2), over-
at 100 °C for 5 min and being allowed to cool, the color
weight (BMI = 25–29.9 kg/m2) and obese (BMI ≥ 30 kg/
intensity was measured at 540 nm (Worthington 1993).
m2). An earlier study showed a lower cut-off (≥ 23 kg/
The serum α-glucosidase activity was determined using
m2) as a risk factor for insulin resistance and diabetes
5 mM p-nitrophenyl-α-D-glucopyranoside (PNPG) pre-
(Zafari et al. 2018). It can be deduced that the subjects
pared in 0.1 M phosphate buffer (pH 6.9) as the substrate,
were majorly overweight and obese. About 42% of the
using the method of Apostolidis et al. (2007).
subjects (17 persons) were overweight, 40% (16 persons)
were obese while 17% (7 subjects) were at border line of
Olagunju et al. Food Production, Processing and Nutrition (2024) 6:32 Page 7 of 17

Table 4 Basic characteristics and anthropometric measurements of study participants

Variables Diabetic (n = 40) Non-diabetic (n = 10) P-value

Gender
Male 16 5
Female 24 5
Age 65.87 ± 9.69 44.70 ± 4.94 0.9898
Anthropometrics
Weight (kg) 73.15 ± 12.50 63.10 ± 8.45 0.3823
Height (m) 1.64 ± 0.07 1.61 ± 0.11 0.9999
Height ­(m2) 2.68 ± 0.22 2.44 ± 0.18a 0.9999 NS
BMI (kg/m2) 28.36 ± 4.65 21.56 ± 1.32 0.0475
Blood pressure
SBP (mmHg) 169.15 ± 20.02 102.33 ± 25.11 0.009
DBP (mmHg) 86.55 ± 6.60 68.73 ± 12.56 0.01
Fasting blood glucose (mg/dL) 117.85 ± 51.66 117.00 ± 9.87 0.05
Fasting insulin (mlU/mL) 47.10 ± 1.60 21.66 ± 2.15 0.002
BMI Body mass index, SBP Systolic blood pressure, DBP Diastolic blood pressure, NS No significant difference
a
=  < 0.05

normal to overweight presenting BMI of 23.48–24.19 kg/ The fasting blood glucose (FBG) was 195 mg/dL on
m2. This corroborates the report that obesity is a com- average for the diabetic group and 117 mg/dL for the
mon complication in diabetic patients (Yang et al. 2021). normoglycemic group. According to the American Dia-
It may also be a pointer to the fact that diabetes accom- betes Association, the normal to healthy range for fasting
panied by overweight, or obesity may predispose the blood glucose before meals is < 100 mg/dL while levels
patient to other diseases. Thus, diabetic patients should above 130 mg/dL is classified diabetes mellitus range.
consciously consider a weight loss program either follow- FBG is used as an index for screening for diabetes mel-
ing a dietary approach or integrating a routine exercise. litus, from existing classification and obtained result,
The present finding is similar to a report by Wanvoegbe FBG of 195 mg/dL is indicative of the diabetic status of
et al. (2016) where 41% of the diabetic subjects were the study participants. Although, majority of the sub-
obese while 39% were overweight. Comparing with jects had FBG averaging 104–119 mg/dL but some oth-
another study by Lucha-Lopez et al. (2021), the BMIs ers had very high FBG up to 230 mg/dL which impacted
recorded were significantly lower than the mean values the overall average reported in the study. Nonetheless, on
reported for both physically active and sedentary diabetic the average, the FBG of the subjects was approximately
Spanish women (34.2 and 38.9 kg/m2 respectively). 137 mg/dL when subjects with extremely high values
The data showed that the non-diabetic subjects had a were excluded. As it stands, the average FBG falls within
good control of their blood pressure exhibiting normal the range reported by ADA (2020) for diabetes mellitus.
blood pressure reading averaged 102/69 mmHg except Fasting insulin level is used as a sensitive marker for
for one who had a low blood pressure (68/55 mmHg). The the assessment of insulin resistance. The fasting insu-
subject confirmed that overtime she regularly recorded a lin (FI) levels were on the average 22 and 47 mlU/mL
low blood pressure. On the contrary, the diabetic subjects for non-diabetic and diabetic (overweight/obese) sub-
showed a poor blood pressure control with an average jects respectively, the FI for the non-diabetic group falls
systolic blood pressure above 150 mmHg and diastolic within the normal range whereas, the FI in diabetic sub-
blood pressure above 80 mmHg. This result agrees with jects was higher. The value obtained (> 30 mlU/mL) may
the reports that diabetes and hypertension have strong indicate insulin resistance in a diabetic individual than
epidemiological association and are considered common in a normoglycemic individual. This is because simi-
comorbidities (Petrie et al. 2018). The comorbidity of dia- lar insulin level is unlikely to comparatively suppress
betes and hypertension may have been promoted by the serum glucose in non-diabetic individual at the same
overweight/obese status; improper management of these rate in diabetic patient. The overweight and obese sta-
disease conditions may promote risk of other cardio- tus in diabetic participants may be implicated to cause
vascular complications such as heart failure, stroke and an impaired cell response to insulin. Regarding the
nephropathy. results of the present finding, the fasting serum insulin
Olagunju et al. Food Production, Processing and Nutrition (2024) 6:32 Page 8 of 17

in diabetic participants was higher and doubles those (protein) and 25.06 g/100 g (fat) recorded for the mar-
of normal weight non-diabetic subjects which may be a ket control sample (COB), it suffices to say that the
consequence of an impairment in β-cell function in dia- nutritional composition of the snack product may have
betic individuals. contributed to the observed postprandial blood glucose
Overall, the prevalence of overweight to obese sta- response.
tus, high fasting blood glucose, high blood pressure and The incremental area under the curve (iAUC) from
abnormal fasting insulin levels observed in diabetic par- postprandial blood glucose reading for diabetic and
ticipants show the subjects exhibit risk factors for cardio- non-diabetic subjects are presented in Fig. 1c. The dia-
vascular diseases. betic group exhibited high iUAC, G1 (18,378 mg.min/
The blood glucose response of healthy non-diabetic dL) was significantly higher than the values for G2 and
subjects who consumed the test samples were recorded G3 (19,258 and 19,852 mg.min/dL respectively) whereas,
over a 2 h period (Fig. 1a). The blood glucose concen- the non-diabetic group had lower values ranged 10,990
tration at start of experiment (0 min) was recorded and to 15,990 mg.min/dL. The postprandial blood glucose
averaged 99.44 ± 14.35 mg/dL. This is in accordance response of diabetic subjects to the multigrain snack
with the normal range (< 100 mg/dL) set for a healthy showed that significant differences were observed after
subject (American Diabetes Association). After inges- 15 min and greater differences after 30 and 60 min of
tion of the snack products, the blood glucose con- test food consumption (Table 5). The group that con-
centration increased for the different groups with a sumed MBY gave a peak glucose reading of 174 mg/dL
peak value observed at 30 min followed by a progres- at 30 min from a 142 mg/dL baseline and a final 133 mg/
sive decline till the end of the 2 h study duration. For dL at 120 min. Group 3 peaked at 60 min (169 mg/dL),
the diabetic group, the experimental snack products from a baseline of 135 mg/dL likewise, G4 peaked at
(MBY and MBZ) exhibited low mean postprandial glu- 60 min (176 mg/dL) from baseline of 125 mg/dL to 2 h
cose level compared to control sample (COB). On one postprandial blood glucose of 157 mg/dL. The blood glu-
hand, the group that consumed MBY (G2) exhibited the cose reading of G3 and G4 increased steadily for the first
highest postprandial glucose response within the first 1 h followed by a marginal decline of 16.49 and 10.49%
15 min (90.67 to 108.67 mg/dL) however, produced the respectively while MBY promoted a 23.92% postprandial
most significant postprandial reduction in glucose level decline. Overall, despite the test meal having the same
(33%). This may be associated with the characteristic amount of available carbohydrates (50 g), the developed
functional composition of the snack product (high ama- multigrain snack showed stable postprandial blood glu-
ranth, protein, and fiber contents). On the other hand, cose which was lower than the high postprandial blood
G3 (group that consumed MBZ) took a longer time concentration recorded in the control sample group (G4).
(30 min) to attain peak postprandial glucose and exhib- The developed snack bar from underutilized conven-
ited a lower increase in blood glucose concentration tional whole grains showed lower blood glucose response
(13%) compared to MBY. The group that consumed the than commercial snack bar products. Other studies have
control sample (G4) exhibited a 23% increase in blood been carried out to develop snack products with blood
glucose after 30 min and a final 18.69% decline in post- glucose control ability for DM patients and healthy
prandial glucose after 2 h of consuming product. The individuals (Manthou et al. 2014; Stamataki et al. 2016;
subjects’ 2 h postprandial blood glucose levels of sub- Yang et al. 2021). The snack products developed in those
jects for MBY are within the normal limits of < 140 mg/ studies also relatively elicited low postprandial glucose
dL (Fig. 1b) thus, suggesting the snack product exhib- response.
ited normal glucose response (Rolfes et al. 2018). MBZ Generally, whole grain cereals are rich in dietary fibre
on the other hand, resulted in a postprandial glucose and resistant starch hence, they are considered a relevant
of 141 mg/dL which is fairly within the specified nor- material for diabetic patients. In addition, high-protein
mal range. However, consumption of control sample plant foods promote satiety and fullness which can be
(COB) as seen in G4 data elicited a postprandial blood useful in suppressing appetite. Therefore, the consump-
glucose of 157 mg/dL which is suggestive of impaired tion of high-protein meals is adjudged to regulate blood
glucose tolerance by subjects. Statistical optimization glucose concentration, hence, may be a safe product for
of preliminarily processed whole grains (amaranth, diabetic patients (Russell et al. 2016). It may not be out
acha, pearl millet) enhanced the protein content of the of place to project that the novel snack product would be
snack products (21.95 and 17.75 g/100 g for MBY and relevant for diabetes control owing to its potential to pro-
MBZ respectively) while the fat content was 17.86 and vide valuable nutrients, promote satiety and slow down
13.55 g/100 g respectively (Table 3). These values are glycemic release.
significantly (p < 0.05) different from the 3.54 g/100 g
Olagunju et al. Food Production, Processing and Nutrition (2024) 6:32 Page 9 of 17

Fig. 1 a Blood glucose concentration in non-diabetic subjects. b Blood glucose concentration in diabetic subjects. c Incremental area
under the glucose curve of study participants. Lines represent mean ± SEM (n = 10). NC: non-diabetic healthy group (positive control); G1: subjects
that did not receive any test food (negative control); G2: subjects fed MBY (snack comprising 90% amaranth, 5% acha, 5% pearl millet); G3: subjects
fed MBZ (snack comprising 47.98% amaranth, 26.68% acha, 25.34% pearl millet); G4: subjects fed COB (commercial oat snack)
Olagunju et al. Food Production, Processing and Nutrition (2024) 6:32 Page 10 of 17

Table 5 Changes in blood glucose concentration after consumption of whole grain developed snack
Time (mins) NC G1 G2 G3 G4

0 86.08 ± 20.15d 133.45 ± 7.68b 142.08 ± 13.26a 134.64 ± 6.94b 125.36 ± 12.97c
d c ab a
15 90.35 ± 9.86 135.79 ± 13.20 156.92 ± 7.64 159.20 ± 8.50 150.45 ± 28.30b
d c a b
30 98.29 ± 10.13 130.63 ± 15.52 174.61 ± 4.49 167.73 ± 7.39 168.36 ± 16.21b
e d c b
60 105.00 ± 7.65 130.98 ± 13.63 155.85 ± 5.95 169.27 ± 9.43 175.91 ± 13.63a
e d c b
90 103.42 ± 9.20 132.26 ± 9.85 145.38 ± 12.09 162.91 ± 8.53 174.27 ± 25.93a
d c c b
120 95.60 ± 11.12 130.11 ± 10.93 132.85 ± 4.31 141.36 ± 7.85 157.45 ± 20.49a
Values are presented as Mean ± SD; Values with different superscripts on the same row are significantly different (P˂ 0.05)
NC Non-diabetic healthy subject in a fasted state, G1 Subjects that did not receive any test food (CTRL), G2 Subjects fed MBY (snack comprising 90% amaranth, 5%
acha, 5% pearl millet), G3 Subjects fed MBZ (snack comprising 47.98% amaranth, 26.68% acha, 25.34% pearl millet), G4 Subjects fed COB (commercial oat snack)

Table 6 Glycemic index and glycemic of load multigrain snack bars in type 2 diabetic and non-diabetic subjects
Samples CHO/100 g Portion size (g) CHO/portion (g) Glycemic Glycemic Load Glycemic Glycemic
Index (GI) (GL) Index (GI) Load (GL)

MBY 59.23a 84.4a 49.99a 52.19c 23.46c 42.77c 12.67c

a a a b a b
MBZ 60.86 82.3 50.03 54.69 25.60 45.03 12.81b
b a a a b a
COB 56.49 88.6 50.02 56.38 23.87 62.24 31.13a
Values represent mean of data set; values with different superscripts on the same column are significantly different (P˂ 0.05)
MBY Snacks bar 1 (snack comprising 90% amaranth, 5% acha, 5% pearl millet), MBZ (snack comprising 47.98% amaranth, 26.68% acha, 25.34% pearl millet); COB Oat
snack bar control (control), CHO Carbohydrate

The glycemic index (GI) of snack bar products ranged to sucrose with high GI; a notable sweetener in commer-
between 52 and 56 while glycemic load (GL) ranged btw cial snack bars (brown sugar) or control (white sugar)
23 and 26 for diabetic subjects (Table 6). However, for which is sucrose with high GI (Alkaabi et al. 2011; Alal-
non-diabetic subjects, the GI obtained in response to wan et al. 2020). Hence, regular consumption of devel-
consumption of a serving of protein-rich snack was 43 oped multigrain snack products may depress the surge
and 45 for MBY and MBZ respectively, COB showed GI in postprandial blood glucose as such pivotal in reduc-
of 62 while GL for the formulated snacks was approxi- ing the risk of type 2 diabetes, cardiovascular disease
mately 13 and for COB, GL was 31. The international and its accompanying complications. Alkurd et al. (2020)
classification of GI is as follows: ≤ 55 (low), 56 to 69 reported intermediate GI (58.94) for multigrain bread
(intermediate), or ≥ 70 (high). The developed snack from whole wheat and different grains. The approved gly-
products had low glycemic indices whereas the commer- cemic load (GL) classification is < 10 for low, 11–19 rep-
cial sample (COB) possessed an intermediate GI for both resents moderate, and > 20 is high. From our result, GL of
groups. Low GI of snacks is indicative of slow release of subjects ranged from 23.46 to 25.60 thus, the snack had a
glucose upon digestion of food thereby controlling glu- high GL. Although, diets low in GI and GL are relevant
cose release into the blood. This corroborates the result to the prevention and management of diabetes, the GL
of blood glucose response (Table 5) wherein slow post- may be significantly reduced by increasing the nutritional
prandial glucose release was observed especially for G2 components of the snack such as the protein and or fibre
who ingested MBY. As is known, several characteristics contents likewise reduction of the portion size per serv-
of food influence its glycemic response; these charac- ing (Brand-Miller 2003).α-amylase and α-glucosidase are
teristics include the quantity and type of carbohydrates, key enzymes responsible for the breakdown of starch.
presence of other nutrients (such as protein, fat, fibre). The former functions to hydrolyze internal α-1,4 glyco-
Hence, the low GI of the multigrain snack bars may be sidic bonds, resulting in the production of maltose and
associated with the low carbohydrate content, the high oligosaccharides whereas, the latter is a crucial enzyme
protein and fibre contents (Table 3) as well as the qual- responsible for catalyzing the final stage of carbohydrate
ity of carbohydrates in the major amaranth grain (Dur- digestion (Kashtoh & Baek 2022). The highest concentra-
gadevi & Nazni 2012; Schneider et al. 2015). In addition, tions of amylase are found in the pancreas and salivary
the sweetener in the developed snack bar is fruit sugar glands, although, equally abundant in other organs (Nater
(fructose); which is reported to have low GI compared et al. 2015). Pancreatic amylase enters the bloodstream
Olagunju et al. Food Production, Processing and Nutrition (2024) 6:32 Page 11 of 17

Fig. 2 a α-amylase. b α-glucosidase activities in serum of experimental subjects. Bars represent mean ± SEM (n = 10). Values are statistically different
at φp < 0.05, φφp < 0.01, φφφp < 0.001 versus NC and λp < 0.05, λλp < 0.01 versus G1. NC: non-diabetic healthy group (positive control); G1: subjects
that did not receive any test food (negative control); G2: subjects fed MBY (snack comprising 90% amaranth, 5% acha, 5% pearl millet); G3: subjects
fed MBZ (snack comprising 47.98% amaranth, 26.68% acha, 25.34% pearl millet); G4: subjects fed COB (commercial oat snack)

and can easily be measured in the blood (Pieper-Bigelow of acute pancreatitis in the subjects. Nevertheless, groups
et al. 1990). High amylase activity in serum could be an that consumed formulated snacks (MBY and MBZ)
indicator of a pancreatic disorder especially acute pan- exhibited 24.73 and 21.72% reduction, respectively. Wor-
creatitis and there exists an established relationship thy of note is the fact that there was no significant dif-
between diabetes and pancreatitis as the former is an ference in the α-amylase activity among the groups fed
etiology to the latter (Hart et al. 2016; Wynne et al. 2019; with the multigrain products, but significantly differed (P
Richardson & Park 2021). Normal serum α-amylase value = 0.0034) from G1 which served as negative control
ranges from 10–150 U/l (Zhuang et al. 2016). According with no dietary treatment. This suggests that consistent
to Fig. 2a, the total serum amylase activity in healthy sub- consumption of the novel snack may promote a signifi-
jects was low (85 U/l) which is within the normal range. cant reduction in the enzyme. Serum α-glucosidase activ-
However, significantly high activity was observed in dia- ity showed similar results although, there was significant
betic subjects (283 U/l), which is a potential biomarker difference among the groups (P value = 0.0037) however,
Olagunju et al. Food Production, Processing and Nutrition (2024) 6:32 Page 12 of 17

no significant difference between diabetic groups and observed in G2 and G3 groups, the groups showed rela-
group that consumed COB. The potency of most diabetic tive comparisons between groups with a relative P value
drugs is as a result of their α-glucosidase inhibitory activ- of 0.006. The increased serum CAT activity by the experi-
ity as the enzyme inhibitor happens to be the most effec- mental groups especially G2 is suggestive of the ability of
tive in reducing post-prandial hyperglycemia (Hossain the snack to promote cellular defense against oxidative
et al. 2020). α-glucosidase inhibitors function to delay damage.
glucose absorption by decreasing carbohydrate digestion NC group had a high SOD activity (95.24 U/l) inferring
and indirectly decreasing insulin surge. Figure 1b shows the availability of sufficient activities of SOD enzyme in
that the NC group had the lowest α-glucosidase activity the non-diabetic healthy individual (Fig. 3b). The SOD
(0.0021 mol/mg protein) while G1 and G4 had the high- enzyme is responsible for the removal of superoxide
est (0.0048 and 0.0044 mol/mg protein, respectively). radicals which are usually produced during any oxidation
Groups G2 and G3 showed a significant decrease of 35.42 reaction process. However, the G1 and G4 groups showed
and 22.92% respectively in α-glucosidase activity. This is a reduction in the activities of this enzyme (35 and 42%
suggestive that the snack products’ carbohydrate diges- respectively) suggesting that substantial concentrations
tion may be delayed, conferring a positive effect on glu- may have been used up in the process of combating oxi-
cose absorption. dative stress as a result of the diabetic condition. G2 and
DM is characterized by persistent hyperglycemia; the G3 groups on the other hand showed upregulated levels
condition that promotes biochemical alterations in glu- of the enzyme (up to 115 and 87 U/l respectively) activity.
cose and lipid peroxidation. Chronic diabetic condition Although, there was no significant difference between the
promotes impaired compensatory response to the antiox- study groups and normal control but, a significant differ-
idant system or increased generation of free radicals thus, ence within the group existed (P value = 0.005).
diabetic subjects may have a compromised antioxidant GSH is an important antioxidant with relevance in
defense system. Serum antioxidant status is an impor- protecting cells against oxidative damage and eventually
tant indicator in the control and management of diabetes chronic disease. As seen for other antioxidant enzymes,
owing to its role in it plays in the protection against free the NC presented the highest GSH level (1.62 mg/g pro-
radicals and oxidative stress. Catalase (CAT), superoxide tein) while G1 had the least level (0.36 mg/g protein) of
dismutase (SOD) and glutathione peroxidase (GPx) are GSH content (Fig. 3c). Interestingly, there was no sig-
enzymatic antioxidants while reduced glutathione (GSH) nificant difference in the levels of GSH content (0.70 to
is a non-enzymatic antioxidant; all these biomolecules 0.82 mg/g protein) within the experimental groups (G2,
play important synergistic role as a defense mechanism G3, G4) whereas, significantly different from NC and G1
from free radicals thereby protecting living cells/organs as the former had very high GSH level while the latter had
from oxidative damage. The results for antioxidant very low levels (P value = 0.0002). GSH can be obtained
enzyme activities are as shown in Fig. 3a-d. from the consumption of foods rich in sulfur-containing
Catalase is an important enzyme responsible for the amino acids moreover, these amino acids are vital for the
neutralization of hydrogen peroxide (­H2O2), a non-rad- maintenance of GSH homeostasis. Grains, however, have
ical reactive oxygen species (ROS), hence, maintaining little glutathione precursor amino acids; this may be the
an optimum level of H ­ 2O2 in the cell. Figure 2a pre- reason for the low GSH levels in the snack-fed group.
sents the serum catalase activity in the different groups Overall, the significantly low levels of each antioxidant
which ranged from 2.28 to 9.92 μmole ­H2O2.consumed/ enzyme in the diabetic subjects as compared to normal
min/mg protein. G1 and G4 showed the lowest activity healthy subjects is a pointer to the prevalence of oxida-
(2.28 and 3.03 μmole ­H2O2.consumed/min/mg protein tive stress and indicative of impaired antioxidant status
respectively). The significantly reduced CAT activity in in diabetic patients. However, the groups that consumed
the G1 group’s serum suggests overwhelming oxidative the experimental multigrain snack exhibited significant
stress as a result of a diabetic condition, thus, indicat- levels of upregulation in the antioxidant profile enzymes.
ing increased production of oxygen-free radicals, which The current finding corroborates the results of Karn et al
could be linked with persistent hyperglycemia, otherwise (2016) in which consumption of a multigrain-formulated
known as high blood glucose (Takemoto et al. 2009). diet exhibited a positive effect on diabetic animals with
The notable low levels of the enzyme in G1 corrobo- respect to antioxidant status.
rates other reports that catalase levels are decreased Diabetes mellitus is associated with increased free radi-
in diabetic subjects compared to non-diabetic subjects cal activity and is characterized by alterations in carbo-
(Ezeiruaku et al. 2016). This may not be unconnected to hydrate, protein, and lipid metabolism. Hyperlipidemia
certain pathophysiologies of the DM disease. Significant is common in patients with type 2 diabetes, the condi-
(p < 0.01, p < 0.001) improvement in CAT activity was tion may induce increased production of lipid peroxides
Olagunju et al. Food Production, Processing and Nutrition (2024) 6:32 Page 13 of 17

Fig. 3 a Catalase activity. b Superoxide dismutase activity. c Glutathione level. d Glutathione peroxidase activity in subject serum. Bars represent
mean ± SEM (n = 10). Values are statistically different at φp < 0.05, φφp < 0.01, φφφp < 0.001, ns versus NC and λp < 0.05, λλp < 0.01, ns versus G1, •p < 0.05,
••
p < 0.01 versus G2. NC: non-diabetic healthy group (positive control); G1: subjects that did not receive any test food (negative control); G2: subjects
fed MBY (snack comprising 90% amaranth, 5% acha, 5% pearl millet); G3: subjects fed MBZ (snack comprising 47.98% amaranth, 26.68% acha,
25.34% pearl millet); G4: subjects fed COB (commercial oat snack), ns: no significant difference

which is an indication of a decline in the cellular antioxi- a multigrain experimental diet, however, promoted a
dant defense mechanism. Lipid peroxides were measured reduction (47% for MBY, 28% for MBZ) in the TBARS
as thiobarbituric acid reactive substances (TBARS). The level which showed no significant difference from the NC
TBARS level in the serum of subjects was found to be group.
significantly (P < 0.05) elevated in G1 (diabetic untreated Significant (P < 0.01) insulin spikes (up to 78 mIU/mL)
group) compared to control subjects (Fig. 4). TBARS were observed in diabetic subjects (Fig. 5). These may be
levels increased twofold (0.36 mmol/mg protein) in dia- a consequent result of type 2 diabetes condition associ-
betics compared to healthy controls (0.15 mmol/mg ated with the buildup of glucose in the bloodstream. This
protein). The high lipid peroxidation in diabetic sub- is suggestive of hyperinsulinemia which is a risk factor
jects evidenced by elevated TBARS may exacerbate the pivotal for promoting insulin resistance and the develop-
occurrence of vascular complications. The ingestion of ment of type 2 diabetes (Janssen 2021). The established
Olagunju et al. Food Production, Processing and Nutrition (2024) 6:32 Page 14 of 17

Fig. 4 Thiobarbituric acid reactive substance levels in serum of human subjects. Bars represent mean ± SEM (n = 10). Values are statistically different
at φp < 0.05 versus NC. NC: non-diabetic healthy group (positive control); G1: subjects that did not receive any test food (negative control); G2:
subjects fed MBY (snack comprising 90% amaranth, 5% acha, 5% pearl millet); G3: subjects fed MBZ (snack comprising 47.98% amaranth, 26.68%
acha, 25.34% pearl millet); G4: subjects fed COB (commercial oat snack)

Fig. 5 Insulin levels in serum of human subjects. Bars represent mean ± SEM (n = 10). Values are statistically different at φφp < 0.01, φφφp < 0.001,
λλ
p < 0.01, ns versus G1, ••p < 0.01 versus G2. NC: non-diabetic healthy group (positive control); G1: subjects that did not receive any test food
(negative control); G2: subjects fed MBY (snack comprising 90% amaranth, 5% acha, 5% pearl millet); G3: subjects fed MBZ (snack comprising
47.98% amaranth, 26.68% acha, 25.34% pearl millet); G4: subjects fed COB (commercial oat snack), ns: no significant difference
Olagunju et al. Food Production, Processing and Nutrition (2024) 6:32 Page 15 of 17

overweight and obese status of the participants (diabetic Funding

This research did not receive any funding from the public, private nor govern-
subjects) as depicted by the BMI (Table 4) is a pointer to ment agency.
the correlation of obesity with hyperinsulinemia/insu-
lin resistance. Figure 4 showed that the consumption of Availability of data and materials
All datasets used and/or analyzed during the current study are available from
the protein-rich, low GI multigrain snack regulated the the corresponding author on reasonable request.
secretion of insulin in diabetic subjects. The decreased
serum insulin levels in groups that consumed test meals Declarations
may be attributed to the low glycemic index of the snack
product thus, offering a lower prevalence of metabolic Ethics approval and consent to participate
The subjects consented to participate in the study, the clinical study was car-
effects. This may suggest that the multigrain snack did ried out following guidelines for human studies with the approval of the Ondo
not rapidly increase the blood glucose levels of the sub- State Health Research Ethics Committee (OSHREC), Ondo State (OSHREC
jects especially sample MBY comprising 90% amaranth, 22/3/2021/316). We hereby certify that the study was performed in accord-
ance with the 1964 Helsinki Declaration and comparable ethical standards. In
5% acha, and 5% pearl millet as G2 showed the lowest addition, the clinical study was conducted in accordance with the guidelines
insulin level. The high content of dietary fiber in ama- as outlined by the Ethics Committee of the School of Agriculture and Agricul-
ranth grain may have contributed to this outcome. This tural Technology, Federal University of Technology, Akure, Ondo State, Nigeria
(approval number FUTA/SAAT/2021/024).
result corroborates an earlier report that a low glycemic
index meal decreases insulin resistance and insulin lev- Consent for publication
els (Gao et al. 2019). Similar to our findings, Sobhana Not Applicable.
et al (2020) reported that consumption of multigrain roti Competing interests
promoted reduced serum insulin in type 2 diabetic par- The authors declare that they have no competing interests.
ticipants. G2 and G3 exhibited postprandial insulin con-
Author details
centrations averaging 43 and 65 mIU/mL respectively. 1
Department of Food Science & Technology, Federal University of Technology,
The level of insulin may be sufficient to effectively remove Akure, Nigeria. 2 Department of Biomedical Technology, Federal University
glucose from the bloodstream. of Technology, Akure, Nigeria. 3 BloomMak Scientific Services, Gbagada, Lagos,
Nigeria. 4 University of Medical Sciences Teaching Hospital, Akure, Ondo State,
Nigeria.
Conclusions
Varied reports have shown that there is an existing lim- Received: 30 March 2023 Accepted: 30 May 2023
itation in the diversity and suitability of snack products
for diabetic patients. The developed high protein, low
glycemic index snack product is a convenient nutri-
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