Accepted Manuscript

Title: Optimization of a fermented pumpkin-based beverage to

improve Lactobacillus mali survival and ␣-glucosidase
inhibitory activity: A response surface methodology approach

Authors: W.Y. Koh, U. Uthumporn, A. Rosma, R. Irfan, Y.H.

Park

PII: S2213-4530(17)30075-7
DOI: https://doi.org/10.1016/j.fshw.2017.11.001
Reference: FSHW 119

To appear in:

Received date: 11-5-2017

Revised date: 30-10-2017
Accepted date: 1-11-2017

Please cite this article as: { https://doi.org/

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Optimization of a Fermented Pumpkin-based Beverage to Improve Lactobacillus mali

Survival and α-Glucosidase Inhibitory Activity: A Response Surface Methodology

Approach

Koh, W. Y.1 Uthumporn, U.1* Rosma, A.2 Irfan, R.3 & Park, Y. H.3

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1
Food Technology Division, School of Industrial Technology, Universiti Sains Malaysia,

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Minden 11800, Penang, Malaysia

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2
Bioprocess Division, School of Industrial Technology, Universiti Sains Malaysia, Minden

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11800, Penang, Malaysia
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Department of Applied Microbiology and Biotechnology, Yeungnam University, Gyeongsan,

South Korea. U
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*Corresponding author
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Uthumporn Utra

Address: Food Technology Division, School of Industrial Technology, Universiti Sains

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Malaysia, Minden 11800, Penang, Malaysia

Tel: 604-6532220
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Fax: 604-6573678

E-mail address: sapina@usm.my

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 R I
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Lactobacillus mali

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Highlights

 Optimum condition to produce a fermented pumpkin-based beverage with anti-

hyperglycemic effect was achieved.

 The fermented product achieved high survival of probiotics, excellent sensory

acceptability, and stable α-glucosidase inhibitory activity.

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Abstract

The aim of this research was to develop an optimum fermentation and composition

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model for a new fermented pumpkin-based beverage with high probiotic survival and α-

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glucosidase inhibitory activity. Relationship between fermentation temperature, inoculum and

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ingredient concentration with response variables (fermentation time at the fermentation
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endpoint pH 4.5, survival rate of Lactobacillus mali K8 in pumpkin-based beverage treated
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with simulated gastrointestinal tract enzyme fluids, α-glucosidase inhibitory activity and
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sensory overall acceptability after 4 weeks of refrigerated storage) was investigated using

response surface methodology. Optimal formulation was obtained at an approximation of

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40 % pumpkin puree concentration, 8 Log CFU/mL inoculum and at 35 ◦C. The product

derived from this optimum formula reached the fermentation endpoint after 28.34 ± 0.10
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hours and the quality changes during 4 weeks storage was studied. The product achieved
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88.56 ± 0.67 % of L. mali survival after treatment with simulated gastric and intestinal juices;
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demonstrated 95.89 ± 0.30 % α-glucosidase inhibitory activity, as well as scored 6.99 ± 0.40

on sensory overall acceptability after 4 weeks of storage. These findings illustrated that the
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model is effective in improving probiotic survival and α-glucosidase inhibitory activity with

excellent sensory acceptability, thus may offer a dietary means for the management of

hyperglycaemia.
Keywords

Probiotics; Response surface methodology; Box-Behnken, Hyperglycaemia; Functional Food

1. Introduction

Type-2 diabetes mellitus is a long term endocrine and metabolic disorder diseases featured

by persistent hyperglycaemia. This might lead to multiple complications and can cause

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cardiovascular disease. In Type 2 diabetes, there are difficulties for insulin to stimulate

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glucose uptake by skeletal muscle, fat and liver cells from the blood [1].

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One of the therapy towards alleviating Type 2 diabetes is focused on promoting the

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efficacy of the body achieving optimal blood glucose after meal. Inhibitors of α-glucosidase,

which assembles the disintegration of disaccharide into glucose, are plausible in impeding

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glucose absorption. Therefore, inhibition of α-glucosidase is contemplated to be a potent
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strategy for the hyperglycemia management. The common synthetic antidiabetic drugs, such
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as meglitinides, biguanides, thiazolidinediones and sulfonylureas, are costly and might also
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have significant side effects such as gastrointestinal disturbance and weight gain. For this
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reason, researchers have shift focus in search for more adequate and safer inhibitors of anti-

hyperglycemic product from natural substances which reportedly has lesser side effects [2].
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Pumpkins (Cucurbita pepo) belong to the family of Cucurbitaceae. Pumpkin flesh

contains a variety of phenolic compounds, flavonoids, vitamins, as well as minerals. It also

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has low calorie content (17 kcal/100g flesh) [3]. One of the major carotenoid in pumpkin
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fruits (>80%) is β-carotene which contributes to the high nutritional value of pumpkins [4].

Traditionally pumpkins are cooked in variety of dishes or used to make desserts and
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beverages. However, pumpkins contain high insulin-dependent sugars level which is

problematic for diabetic patients [5]. Therefore, in this study, technology such as

fermentation is utilized in the development of pumpkin based beverage in which the

microorganisms could utilize the sugar during fermentation. This reduces the insulin-

dependent sugars so that it is more suitable for the consumption of diabetic patients.

Moreover, fermentation of vegetables and fruits such as pumpkins may not only improve its

food safety levels and shelf life, but may also enhance the availability of certain nutrients [6].

According to FAO/WHO in 2002 [7] definition, ‘probiotics are live microorganisms

that confer health benefits to host when administered in adequate amounts’. There is a

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growing demand for dairy alternatives by cause of lactose intolerance, milk protein and

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cholesterol allergic, stimulating development of non-dairy probiotic foodstuff [8]. Probiotic

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beverages which are fruit and vegetable based are non-dairy and are viewed positively by the

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public with regards to health and have becoming one of the main fruit and vegetable intake

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methods [9]. Several probiotic beverages based on fruits and vegetables have been studied
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including artichoke [10], pineapple [11], carrot [12], broccoli and cherry [13].
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Probiotic microorganisms must have high survivability through the gastrointestinal
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tract which is harsh for most microorganisms such as to tolerate acid, bile and gastrointestinal

enzymes, eventually attached to and set up colonies in the intestinal epithelium in order to
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improve the host’s health [14]. Pumpkin flesh have the appropriate matrix with pH value

more than five and also adequate level of oxygen for the probiotic bacteria to survive long
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enough over gastric passage, where the matrix’s buffering capability increases the gastric

tract’s pH and further stabilize as well as provide protection to the probiotics [15]. From this
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point of view, pumpkin could serve as a delivery carrier for sufficient viable probiotics to

assure active healthy benefits to the host.

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In previous studies (Koh, W. Y., Uthumporn, U., and Rosma, A., unpublished work)

we have demonstrated that Lactobacillus mali K8 isolated from kefir grains exhibited high α-

glucosidase inhibition activity. In the present research, L. mali K8 was used as an inoculum

for the fermentation of a pumpkin-based beverage in order to further explore the concept of
pumpkin fruits could serve as a delivery vehicle for probiotics. The primary objectives of this

study were (1) to optimize the variables i.e. pumpkin puree concentration, fermentation

temperature and inoculum concentration levels for the development of a fermented pumpkin-

based beverage that is stable for 4 weeks of cold storage, (2) to establish the relationship

among the aforementioned variables with fermentation time, the survival of probiotics in

fermented pumpkin-based beverage after treated with simulated gastrointestinal fluids, α-

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glucosidase inhibitory activity and sensory overall acceptability after 4 weeks of cold storage.

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Response surface methodology was employed to accomplish these objectives so that the anti-

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hyperglycemic functional beverage product can provide convenient probiotics and nutrients

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intake to the general public, which may better manage hyperglycemic condition in Type-2

diabetes patients.
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2. Materials and methods
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2.1. Preparation of inoculum
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Lactobacillus mali K8 (isolated from kefir grains) was used for the pumpkin-based
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beverage’s fermentation. The cultures were first grown in sterile Lactobacillus de Man

Rogosa Sharpe broth, incubated (48 h, 37 ◦C) and then centrifuged (14, 310 × g, 20 min).
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Subsequently, the pellets were suspended in sterilized saline solutions and re-centrifuged with

the aforementioned settings. The washed cells were then adjusted in sterilized saline to three
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different concentrations which are 6 Log colony forming units per mL (CFU/mL), 7 Log

CFU/mL, and 8 Log CFU/mL, respectively. Each of these suspension concentrations were
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employed at 1 mL/100 mL across the different fermentation series.

2.2. Preparation of pumpkin based beverage

Figure 1 outlined the preparation for the pumpkin based beverage. Freshly collected

pumpkins were washed thoroughly with chlorinated water (50 ppm sodium hypochlorite) and
the fruits were diced into cubes. The pumpkin cubes was steamed at 100 ◦C for 20 min in a

laboratory autoclave to soften the flesh so that they can be easily homogenized into puree.

The pumpkin puree and distilled water were measured according to the formulations

generated by response surface methodology coupled with Box-Behnken Design in Design

Expert ® (Table 2) and mixed in 250 ml Erlenmeyer flask. The pumpkin juice was

autoclaved (121 ◦C, 20 min) and cooled down before L. mali K8 inoculum was added at 1

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mL/100 mL and fermented according to the independent parameters as in Table 2. The pH

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value of the pumpkin based beverage was monitored every hour using a pH meter (Mettler-

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Toledo, Switzerland) with a glass electrode at room temperatures throughout the fermentation.

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The time required to decrease to pH4.5 was recorded as the fermentation time (TpH4.5) based

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on pH 4.5 being set as the fermentation endpoint in probiotic beverage production [16].

Finally, the end beverage products were kept at 4 ◦C prior to use.

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2.4. Survival of L. mali K8 in fermented pumpkin based beverage after incubated in simulated
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gastrointestinal fluids

The fermented products were stored at 4 ◦C for 4 weeks and L. mali K8 survival rates
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at every week of storage after simulated gastrointestinal juices exposure were determined.
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Simulated gastric juice at pH 2 was imitated by preparing hydrochloric acid buffer consisting

of 8 g/l sodium chloride, 0.2 g/l potassium chloride, 8.25 g/l sodium phosphate dibasic
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dehydrate, 14.35 g/l sodium phosphate monobasic, 0.1 g/l calcium chloride, 0.18 g/l
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magnesium chloride hexahydrate and 3 g/l pepsin. The pumpkin based beverage samples (10

mL) was inoculated into 90 mL of simulated gastric juice and was subjected to incubation (37
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◦
C, 3 h) in a rotary shaker.

After incubation, the solution were centrifuged and washed with 0.85% (w/v) pre-

reduced saline solution and added into 9 mL of simulated intestinal juice (pH 7.4) consisting
of 6.50 g/l sodium chloride, 0.84 g/l potassium chloride, 0.22 g/l calcium chloride, 1.39 g/l

sodium bicarbonate, 0.50 g/l L-cysteine hydrochloride, 3 g/l bile salt and 1 g/l pancreatin

(Sigma-Aldrich). Consequently, the solution was incubated with consistent shaking (37 ◦C, 3

h). Subsequently, 1 mL of samples was inoculated to 9 mL of pre-reduced phosphate buffer

(0.1 M, pH 7.4) and was subjected to stomacher homogenization (5 min) and then

centrifugation (10,000 g, 10 min, 4 ◦C). Serial dilution of pellet with saline solution was made

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and spread-plated on MRS agar added with 0.05% (w/v) L-cysteine hydrochloride and was

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subjected to incubation (37 ◦C, 48 h). Viable cells were enumerated and conveyed as Log

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CFU/mL after incubation. The survival rates (%) was determined as (log CFU N1/log CFU

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N0) ×100% where N1 represented the total viable count of the microbial strain in pumpkin

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beverage after treatment whereas the total viable count of the microbial strain in pumpkin
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beverage before treatment was represented by N0. All experiments were performed in
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triplicate [17].
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2.5. α-Glucosidase enzyme inhibitory activity

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Rat intestinal acetone powder (Sigma-Aldrich, I1630) was used as an enzyme source.

Ultra-pure water was used to dissolve the acetone powder at 10 mg/mL and the mixture was
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subjected to centrifugation (16, 000 × g, 30 min, 4 ◦C), and the supernatant was used as the α-

glucosidase solution in this assay. Beverage sample was centrifuged at 5939 g for 10 min at 4
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◦
C. The pH of supernatant was adjusted to pH 7.4 and centrifuged again at 13,362 g for 10
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min at 4 ◦C. The supernatant was then filtered through a 0.22 µm membrane filter before

conducting enzyme inhibition assay [18].

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A total of 100 µL beverage samples (at each storage time, week 0, 1, 2, 3 and 4) or
acarbose (Cayman Chemical) (10 mg/mL) were mixed with 100 µL α-glucosidase enzyme
solution and 100 µL of phosphate buffer (0.1 mol/L, pH 6.9) and then was subjected to pre-
incubation (25 ◦C, 5 min). Consequently, 100 µL of 5 mmol/L p-nitrophenyl-α-D-
glucopyranoside solution was added as substrate after pre-incubation and was subjected to
incubation (25 ◦C, 10 min). After the incubation, α-glucosidase enzyme inhibition was
measured by taking the absorbance at 405nm and the data was calculated using equation
below [19].

where x0, y0, x and y are the absorbance with enzyme and without the sample, absorbance

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without enzyme or the sample, absorbance with enzyme and the sample, and absorbance with

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the sample but without enzyme, respectively.

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2.6. Sensory evaluation

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Sensory properties of pumpkin based beverage were evaluated by a untrained sensory

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panel (n = 50), which was performed immediately after beverages removal from storage
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conditions. Beverages were served in food serving trays with codes. Panelists were asked to
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evaluate appearance (colour), smell, taste, texture (consistency) and overall acceptability of
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the pumpkin based beverages at week 0, 1, 2, 3, and 4 of storage. A seven-point hedonic scale

was used by the panelists to evaluate sensory attributes of experimental pumpkin based
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beverage at each storage time. The limit of acceptance was rated at 2.5 which means that any

attributes evaluated below that score was considered to represent the end of shelf-life [20].
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2.7. Experimental design

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Response Surface Methodology coupled with Box-Behnken design was utilized to

optimize the levels of fermentation temperature and ingredient concentration.

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The range for independent variables i.e. fermentation temperature (X1), inoculum

concentration (X2) and pumpkin puree concentration (X3) were derived from preliminary

experiments results (Table 1). The twelve factorial points and centre points were repeated for

five times. Fermentation time at the fermentation endpoint pH 4.5 (TpH4.5), α-glucosidase
inhibitory activity (α-G), survival of L. mali K8 in pumpkin based beverage treated with

simulated gastrointestinal fluids (SGI) and sensory overall acceptability (SOA) after 4 weeks

of cold storage were selected as combination responses presented in Table 2. Each

experimentation was replicated three times and observed responses were calculated through

their mean values. These experiments were undertaken in a random way to minimize

perceived unexpected variability in the responses.

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The independent variables’ actual values as shown in Table 2 were coded using the

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equation down below:

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(1)

Where x, xi, x0 and Δx are the coded value, corresponding actual value, actual value in the
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centre of the domain and increment of corresponding to a variation of one unit of x,
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respectively.
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The mathematical model corresponding to the composite design is:

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(2)
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Where, y, β0, βi, βii, βij are the predicted response (TpH4.5, α-G, SGI and SOA), constant

coefficient, linear coefficient, quadratic coefficient and cross product coefficient, respectively
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and Xi and Xj are assigned as independent variable while Ꜫ represented as error or noise [21].
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2.8. Statistical analysis

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For the storage quality experiments, all samples were replicated three times and the

data were depicted as mean ± standard deviations. Differences between statistical

significance were evaluated using one-way analysis of variance.

 Design Expert® 10.0.1 were used for data analysis of response surface methodology

experimental design and predicted or estimated responses calculations. The statistical

experiment’s validity was confirmed through additional experiments.

3. Results and discussion

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3.1. Model fitting

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Response surface methodology optimization is more favourable than the single

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parameter optimization in terms of lower cost, shorter time, smaller space and quantity of raw

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material required for experimentation. The adequacy of second order quadratic polynomial

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models describing the effects of three independent variables: pumpkin puree concentration
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(X1), inoculum concentration (X2) and fermentation temperature (X3) on responses (TpH4.5, α-
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G, SGI and SOA after 4 weeks refrigerated storage). A model is considered as well fitted
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with the experimental data when significant regression model (Pm < 0.05) and non-significant

(PIof > 0.05) lack of fit is obtained [22]. As demonstrated in Table 3, all proposed
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mathematical models could be utilized sufficiently to estimate the dependent variables as a

function of the independent variables. At all cases, the statistical models consisted of
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significant determination coefficeints (R2 > 0.98), non-significant P-value for lack of fit
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relevant to pure error (p > 0.05) and low correlation variation % (C.V.%). The adequate
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precision level of all models were well above the threshold value of 4 indicating adequacy of

the signals and model to navigate the design [23].

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The four responses of interest were fermentation time at the fermentation endpoint pH

4.5 (TpH4.5), α-glucosidase inhibitory activity (α-G), survival of L. mali K8 in the beverage

after incubation in simulated gastric and intestinal fluids (SGI), and sensory overall

acceptability (SOA) following 4 weeks storage under refrigeration (4 ◦C) condition. The
outcomes of the 17 experiment runs using the Box-Behnken designs were presented in Table

2. The values for TpH4.5 ranged from 12 to 120 hours, and the maximum point was found

using formulation of X1 40%, X2 7 Log CFU/mL, X3 20 ◦C. The minimum TpH4.5 was

determined in the existence state of experimental conditions of X1 30 % (v/v), X2 8 Log

CFU/mL and X3 40 ◦C. A broad range of values for SGI after 4 weeks refrigerated storage

was also found (survival rate of 18.04 90.86 %). The maximum survival was found under

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the conditions of X1 40 % (v/v), X2 7 Log CFU/mL and X3 40 ◦C. On the other hand,

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α-G values after 4 weeks storage ranged from 10.12 to 95.97 % inhibition, and the greatest

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value was set up in conditions of X1 40 %, X2 8 Log CFU/mL, X3 30 ◦C. The values

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for SOA after 4 weeks storage was ranged widely from 1.89 6.99. The maximum point was

found using experimental conditions of X1 30%, X2

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8 Log CFU/mL, X3 40 ◦C. These
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conditions differ according to the responses, therefore in order to acquire short TpH4.5 coupled
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with high values of α-G, SGI and SOA, an optimum process was examined.
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As demonstrated in Table 3, the outcome of analysis of variance postulated the

quadratic model was significant at each of the responses investigated. Based on the data
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provided in Table 3, second order polynomial equations excluding non-significant (p > 0.05)

factors were provided showing the relationships between the independent variables and
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responses, as shown in Equations (3) (6), respectively.

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TpH4.5 40.80-4.50 X1-4.25 X2-26.00 X3+7.75 X1 X2-22.25 X1 X3 +19.98 -

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14.03 +14.48 (3)

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α-G 54.29+17.59 X1+16.76 X2+10.94 X3+8.12 X1 X2+6.07 X1 X3-3.48 X2 X3-

16.51 +14.60 -4.68 (4)

SGI 65.26+15.91 X1+14.15 X2+12.89 X3 +12.44 X1 X3-4.62 X2 X3-16.33 (5)

SOA 5.31+1.03 X1+1.10 X2+0.31 X3-0.53 X1 X2+1.00 X1 X3+0.75 X2 X3-0.98 +0.35 -

0.68 (6)

3.1.1. Fermentation time

The pH value 4.5 was selected as the fermentation endpoint because it’s safer for the

food products to be stored at this pH value and any food products with pH value above 4.5

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can be conducive for food pathogen growth [11]. As deduced from Table 3, the linear term of

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fermentation temperature (X3) had the largest effect (F value = 26.00) on the TpH4.5 followed

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by the interaction term of pumpkin puree concentration and temperature (X1X3) and quadratic

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term of pumpkin puree concentration ( ). Besides that, the linear term of pumpkin puree

concentration (X1) and inoculum concentration (X2), interaction term of pumpkin puree and

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inoculum concentration (X1X2), and the quadratic terms for inoculum concentration and
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temperature ( , respectively) demonstrated significant values (p < 0.05) (Table 3).
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Nevertheless, the interactions between inoculum concentration and temperature (X2X3) does
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not contribute significantly to the values of TpH4.5. The statistical model demonstrated
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significant coefficient (r2 = 0.9845), and non-significant p-value for lack of fit relevant to

pure error (Plof = 0.9481; p > 0.05). The polynomial Equation (3) fitted the response TpH4.5
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adequately.

3.1.2. α-Glucosidase inhibitory activity

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All the estimated coefficients of the fitted second-order polynomial models were

significant in α-glucosidase inhibitory activity after 4 weeks refrigeration storage response.

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The linear term of pumpkin puree concentration (X1) demonstrated the greatest effect (F

value = 17.59) (Table 3), trailed along by the linear term of inoculum concentration (X2), and

the quadratic term of pumpkin puree concentration . The predicted model in α-

glucosidase inhibitory activity response possessed high coefficient of determination (r2 =

0.9985) and insignificant lack of fit (PIof = 0.2523), indicates well fit to the mathematical

Equation (4).

3.1.3. Survival of L. mali K8 in beverage after incubation in simulated gastric and intestinal

fluids

The quadratic term corresponding to pumpkin puree concentration , with the

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highest F value (16.33), illustrated the greatest contribution (p < 0.05) to the SGI model,

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trailed along by the linear terms of pumpkin puree (X1) and inoculum concentration (X2)

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(Table 3). Additional terms i.e. linear term of fermentation temperature (X3), interaction

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terms corresponding to pumpkin puree concentration and temperature (X1X3), and inoculum

concentration and temperature (X2X3) also demonstrated significant values (p < 0.05) (Table

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3). While, the term corresponding to the interactions pumpkin puree and inoculum
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concentrations (X1X2), the quadratic terms of inoculum concentration and fermentation
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temperature illustrated a negligible effect. The predicted model demonstrated coefficient
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of determination (r2) was 0.9951, whereas lack of fit p-value was 0.9375 which implies a
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good fitting to the Equation (5).

3.1.4. Sensory overall acceptability

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Based on the ANOVA test as shown in Table 3, all independent variables affecting
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the SOA response significantly (p < 0.05). The inoculum concentration in its linear term (X2)
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had the most significant effect on SOA, accompanied by linear term of pumpkin puree

concentration (X1) and interactions between pumpkin puree concentration and temperature
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(X1X3). The predicted model showed coefficient of determination, r2 = 0.9875, with the lack

of fit p-value = 0.6052, which indicates an adequate fitting to the mathematical model

(Equation (6)).
3.2. Response surface model and contour plot interpretation

The regression equations were presented in three-dimensional surface plots and two-

dimensional contour plots. This aid in visualizing the relationship between responses and

each variable’s experimental levels and two test variables’ interactions. Significance of the

variables can be noted through the elliptical or circular form of the contour plots. A saddle or

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elliptical contour plot of response surfaces denotes that there were significant interactions

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between the correlated variables, while a circular contour denotes otherwise [24].

The response surfaces and contour plots produced by the model for TpH4.5, α-G, SGI

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and SOA after 4 weeks of storage (Figs 2-5, respectively) demonstrated the relationship

between independent and dependent variables, two variables were portrayed in one three-

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dimensional surface plot while the other variable were retained at zero. All responses (TpH4.5,
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α-G, SGI and SOA values) were sensitive to slight modifications of the independent variables
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(pumpkin puree concentration, inoculum concentration and fermentation temperature).
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3.2.1. Fermentation time

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The effect of inoculum and pumpkin puree concentration was displayed in a two-

dimensional contour and three-dimensional response surface plot (Figure 2a), which showed
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the factors’ linear and quadratic effects respectively. The increment in pumpkin puree

concentration associated with the decrease in the response of TpH4.5, from 82 to 18 hours.
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However, a continued increase in the pumpkin puree concentration ( 35 to 40 %) produces

an increase in TpH4.5 response. On the other hand, the increment of inoculum concentration
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decreases the TpH4.5 reaching its maximum value at 6.5 Log CFU/mL, followed by a

constant value and then slow decrease.

Figure 2b illustrated the quadratic and linear effects of fermentation temperature and

pumpkin puree concentration variables. The increase in fermentation temperature stimulated

the decrease TpH4.5 response, but continued increase in the fermentation temperature ( 35 to

40 ◦C) induced increase in TpH4.5 response. As can be observed in Figure 2c, there is

negligible interaction between fermentation temperature and inoculum concentration (p >

0.05). Furthermore, there is observable linear effects of fermentation temperature and

inoculum concentration. Figure 2c demonstrated that an increase of fermentation temperature

reduces TpH4.5 values, with the same effect being observed in inoculum and pumpkin puree

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concentration. These findings propose that shorter TpH4.5 could be achieved by increasing

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temperature, inoculum and pumpkin puree concentration.

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3.2.2. α-Glucosidase inhibitory activity

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Figure 3a illustrated inoculum and pumpkin puree concentration’s quadratic effects on

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α-G values after 4 weeks of storage. As inoculum concentration increases up to
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approximately 7.0 Log CFU/mL, α-G increases, but further increase in this variable ( 7.0 to
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7.5 Log CFU/mL) α-G response became plateau and eventually increase again. Figure 3b
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shows the quadratic and linear effects of fermentation temperature and pumpkin puree
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concentration variables. An increase in pumpkin puree concentration stimulated an increase

in the response of α-G reaching its maximum point at 35 %, and then slightly decrease.
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While fermentation temperature and inoculum concentration’s linear effects can be observed

Figure 3c, where the increase of fermentation temperature increases the α-G values. The same
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effect can be observed for inoculum and pumpkin puree concentration as well. The positive
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impact of these parameters propose that higher α-G is achievable by increasing temperature,

inoculum and pumpkin puree concentration.

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3.2.3. Survival of L. mali K8 in beverage after incubation in simulated gastric and intestinal

fluids
 The contour plot in Figure 4a presented a circular shape, indicates insignificant

interaction between inoculum and pumpkin puree concentrations (p > 0.05). Quadratic and

linear effects of temperature and pumpkin puree concentration variables can be observed in

the interaction plot (Figure 4b). In this case, there are significant interaction between the two

variables (p < 0.05). While the rise in temperature increases the SGI value after 4 weeks

storage, the increment of pumpkin puree concentration up to 30%, resulted in the increment

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in the SGI response whereas more elevation in pumpkin puree concentration causes a

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decrease in SGI values. There is also significant interaction between both temperature and

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inoculum concentration (p < 0.05) as observed in Figure 4c. The plot shows that a continuing

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increment of both temperature and inoculum concentration advocates the increase in SGI

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values. These findings postulated that higher SGI could be achieved by increasing
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temperature, inoculum and pumpkin puree concentration.
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3.2.4. Sensory overall acceptability
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Figure 5a-c illustrated the contour and response surface plots for SOA after 4 weeks
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storage. The quadratic and linear effects of inoculum and pumpkin puree concentration on

SOA response can be clearly observed in Figure 5a. Therefore, an elevation in inoculum
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concentration produces an elevation in SOA and the same effect can be observed for pumpkin

puree concentration. As illustrated in Figure 5b, while the increase in temperature increases
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the SOA values, the increase of pumpkin puree concentration from 20 to 30 % resulted in a
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preliminary increase in SOA trailed around with a rapid decrease. The Figure 5c illustrated

the linear and quadratic effects of both temperature and inoculum concentration. The SOA
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score increased as a result of the elevation of temperature reaching its maximum value at 30
◦
C, followed by a constant value and then slow decrease.

3.3. Predictive models verification

 An optimization investigation was performed based on these findings to find the

optimal ingredient concentrations as well as optimal fermentation conditions for individual

and all combined response with the aim to obtain fermented pumpkin-based beverage product

which exhibit short TpH4.5 and high values of α-G, SGI and SOA properties and stable for at

least 4 weeks refrigerated storage.

The optimal conditions for each separate response with the experimental and

T
predicted values were showed in Table 4. Optimal formulation was obtained at an

IP
approximation of 40% pumpkin puree concentration, 8 Log CFU/mL inoculum and at 35 ◦C.

R
The optimal fermentation temperature values for short TpH 4.5, high α-G, SGI and SOA were

SC
within the range of 20-40 ◦C and is consistent with de Castro et al. in 2009 [25], pointing out

that lactic beverage fermented at 40 ◦C achieved maximum viability of lactic acid bacteria.
U
N
Besides that, Filannino et al. in 2015 [13] demonstrated that Lactobacillus spp. fermented in
A
10 % (vol/vol) broccoli puree presented a discerning cells viability. On the other hand,
M

Speranza et al. in 2012 [26] applied 8 Log CFU/mL of probiotic inoculum level for anchovies

fermentation and the fermented product maintained high survival of the tested probiotic
ED

strains without affecting the sensory acceptance.

As can be seen in Table 4, the optimal conditions gave TpH4.5, α-G, SGI and SOA
PT

values of 28.34 hours; 95.89 %; 88.56 %; and 6.99 score, respectively. Results showed that

there is insignificance difference in the predicted and experimental values. Hence, the models
E
CC

acquired in this research could be employed to optimize the fermentation temperature and

ingredient concentrations of pumpkin based beverage to obtain short TpH4.5 and prominent
A

values for useful properties such as α-G and SGI with satisfied SOA after 4 weeks

refrigerated storage.

3.4. Storage quality

 Acarbose is a commercially standard drug available to delay starch and

oligosaccharides degradation, decreasing the absorption rate of glucose and inhibits the

increase in postprandial blood glucose. However abdominal distention, flatulence and

possibly diarrhea are reported as some of its side effects. Searching for safe and effective

inhibitors from natural sources are of emerging interest [19].

The inhibitory effects of the inoculated optimized beverage, non-inoculated pumpkin

T
juice and acarbose on α-glucosidase are shown in Figure 6. α-Glucosidase inhibiting

IP
activities of the samples were in the following order: Acarbose > inoculated optimized

R
beverage > non-inoculated pumpkin juice. High levels of α-glucosidase inhibition (92.03 to

SC
95.89 %) activity for as long as 4 weeks of cold storage observed in L. mali K8 inoculated

optimized beverage is comparable to that in acarbose (98.93 %). α-Glucosidase inhibition of

U
N
the inoculated optimized beverage product gradually increases throughout the storage study,

suggested that K8 strain continued to secrete bioactive components which are potent α-
A
M

glucosidase inhibitors during storage. Our findings showed that the inoculation of L. mali K8

strain did not only enhance but may also increase the pumpkin juice α-glucosidase inhibitory
ED

capacity with storage (Figure 6).

Jin et al. in 2013 [27] have reported pumpkin-rich diets prevent excessive rise of the
PT

blood glucose level after a mixed carbohydrate diet, which might be due to the potential α-

glucosidase inhibition activity of pumpkin polysaccharides in its fruits. Adams et al. in 2011
E
CC

[28] concluded that pumpkin fruit have active hypoglycemic properties and antidiabetic

effects due to its abundance of pectin (dietary fibre) and non-pectin polysaccharies which
A

when consumed is purported to improved glucose tolerance and control glycaemia by

lessening the blood glucose elevation. Therefore, the fermentation of pumpkin juice by the L.

mali K8 bacteria could enhance the α-glucosidase inhibition of pumpkin juice tremendously

due to the generation of oligosaccharides hydrolyzed by the bacteria from polysaccharides in

pumpkin juice [29]. Moreover, naturally occurring plant’s polyphenols in pumpkin juice

could bind to the reactive sites of enzymes and altering its catalytic activity, thus are

considered potent and safe inhibitors of carbohydrate hydrolysing enzymes [30].

Strong inhibition of intestinal α-glucosidase could be a potent strategy for the

management of Type-2 diabetes [31]. Therefore, we suggest that inhibition activity against α-

glucosidase could be part of the possible mechanisms of the optimized beverage in dietary

T
management of diabetes, by retardation of starch hydrolysis in the gastrointestinal tract.

IP
Additionally, no sugar was added in the production of fermented pumpkin based beverage

R
and the non-insulin dependent sugars in pumpkin puree can be fermented by L. mali K8 to

SC
produce metabolites that might improve the functional properties of the beverage and is

suitable for diabetic folklore consumption.

U
N
As illustrated in Figure 7, the survival rates of L. mali K8 in optimized beverage after
A
incubation in simulated gastric and intestinal fluids showed variation of less than 2 %
M

throughout the storage period. The viability of L. mali in optimized beverage during

refrigerated storage period of 4 weeks and after treated in simulated gastrointestinal fluids
ED

was consistently over the minimum recommended level of 6 Log CFU/g implied positive

health potency in the intestines [32].

PT

Du et al. in 2011 [33] reported that there is high resistance of the oligosaccharides

from pumpkin pulp towards hydrolysis in artificial human gastric juice and can stimulate the
E
CC

Lactobacilli growth. In addition, Lactobacilli strains are able to metabolize and adhere to

pumpkin’s polysaccharides [15]. Therefore, we proposed that the polysaccharides and high
A

fiber content in the fermented pumpkin based beverage may acts as a buffer against the acidic

condition in the stomach by binding to excess acids produced by the digestive system

protecting the probiotic viability during refrigerated storage [34]. This increases the survival

rate of the probiotic microorganism in the product during the refrigerated storage. Through
this it is postulated that the pumpkin juice could serve as a suitable food matrix with

polysaccharides promoting microbial survival rates.

On the other hand, refrigeration storage was favorable to the preservation of probiotic

populations in fermented pumpkin based beverage. Similar to what was detected in this

research study for fermented pumpkin based beverage, Shukla et al. in 2012 [35] disclosed

that probiotic L. acidophilus in whey and pineapple beverage maintained at 107 CFU/mL

T
throughout 4 weeks cold storage (4 ◦C); whereas for the probiotic pineapple and whey

IP
beverage stored at 30 °C, the total viable count of L. acidophilus in declined gradually and

R
after 5 days no viability of probiotic cells was observed.

SC
Optimized fermented pumpkin based beverage was able to maintain its sensory

U
attributes for at least 4 weeks under cold storage as shown in Figure 8 as the difference
N
observed between the storage period were insignificant (p > 0.05). The sensory acceptance
A
for the optimized fermented beverage can be accredited to compounds such as free amino
M

acids, exopolysaccharides and interesting volatile compounds (esters, aldehydes, phenyl,

ketones, acids, alcohols, furan and benzothiazole), which were the byproduct of the
ED

fermentation enriching the pumpkin juice [5].

4. Conclusions
PT

Survival rate and α-glucosidase inhibitory activity of Lactobacillus mali K8 in fermented

E

pumpkin-based beverage were maximize. Optimized fermented pumpkin-based beverage

product achieved high sensory acceptance and 95% α-glucosidase inhibitory activity. After
CC

treatment with simulated gastrointestinal fluids and 4 weeks cold storage, L. mali K8
maintained up to 88% survival rate. In conclusion, pumpkin-based beverage has the potential
A

to be used as a probiotic carrier and to cultivate L. mali for the development of a functional
anti-hyperglycemic drink.
Acknowledgments

The authors would like to appreciate and acknowledge ASEAN University Network
(AUN) and Korea Association of the Southeast Asian Studies for providing financial
 support under ASEAN-ROK Academic Exchange Programme 2016/2017; and Universiti
Sains Malaysia (USM) Vice Chancellor Award Scholarship and grant (RUI,
1001/PTEKIND/811339).

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ED
E PT
CC
A
Fig. 1. Flow chart of experiments and processes performed.
Fig. 2. Response surface plots illustrating the interactive effects of (a) Pumpkin puree
concentration vs Inoculum concentration, (b) Pumpkin puree concentration vs Fermentation
temperature, (c) Inoculum concentration vs Fermentation temperature on fermentation time
(pH 4.50 set as the fermentation endpoint) of the fermented pumpkin-based beverage.
Fig. 3. Response surface plots illustrating the interactive effects of (a) Pumpkin puree
concentration vs Inoculum concentration, (b) Pumpkin puree concentration vs Fermentation
temperature, (c) Inoculum concentration vs Fermentation temperature on α-glucosidase

T
inhibitory activity percentage of the fermented pumpkin-based beverage after 4 weeks
refrigerated (4 °C) storage.

IP
Fig. 4. Response surface plots illustrating the interactive effects of (a) Pumpkin puree
concentration vs Inoculum concentration, (b) Pumpkin puree concentration vs Fermentation

R
temperature, (c) Inoculum concentration vs Fermentation temperature on L. mali survival in

SC
the gastrointestinal condition of the fermented pumpkin-based beverage after 4 weeks
refrigerated (4 °C) storage.
Fig. 5. Response surface plots illustrating the interactive effects of (a) Pumpkin puree

U
concentration vs Inoculum concentration, (b) Pumpkin puree concentration vs Fermentation
temperature, (c) Inoculum concentration vs Fermentation temperature on sensory overall
N
acceptability score of the fermented pumpkin-based beverage after 4 weeks refrigerated (4 °C)
storage.
A

Fig. 6. Changes in α-glucosidase inhibition (%) in fermented pumpkin-based beverages

M

(obtained from optimized fermentation condition) during refrigerated (4 °C) storage in

comparison with acarbose (positive control) and non-inoculated pumpkin puree (negative
control). Each value in the table represents the mean ± SD (n = 3). Error bars represent the
ED

standard deviation of all trials. Different superscripts have mean ranks that are significantly
different from each other. Mean values with different uppercase alphabets (A-G) indicate
significantly difference (p < 0.05) among storage period (weeks); mean values with different
PT

lowercase alphabets (a-g) indicate significant difference (p < 0.05) among samples.
Fig. 7. Survival rate (%) of L. mali in the fermented pumpkin-based beverage (obtained from
optimized fermentation condition) during refrigerated (4 °C) storage. Error bars represent the
E

standard deviation of all trials. Different superscripts have mean ranks that are significantly
CC

different from each other. Mean values with different lowercase alphabets (a-g) indicate
significant difference (p < 0.05) between storage period (weeks).
Fig. 8. Seven-point hedonic sensory evaluation of the fermented pumpkin-based beverage
A

(obtained from optimized fermentation condition) stored at 4 °C for 4 weeks.

 Pumpkin

Washing
(Chlorinated water [50ppm NaOCl])

Remove seed and peel, cutting

(Thickness: 2cm, diameter: 3cm [~12.5 g])

T
IP
Autoclave steaming
(100 °C, 20 min)

R
Homogenize

SC
(Mixi grinder)

Distilled water Pumpkin puree

U
N
Autoclave sterilization
A
(121 °C, 20 min)
M

Inoculation (L. mali K8 strain, 10% v/v) and incubation

ED

Fermented pumpkin-based beverage

Determination of fermentation time

PT

(TpH4.5)
E

Storage at 4 °C for 4 weeks

CC

Determination of L. mali survival in gastrointestinal condition, α-glucosidase inhibitory

activity percentage and sensorial acceptability score
A

Fig. 1. Flow chart of experiments and processes performed.

(a) (b)

T
R IP
SC
(c)

U
N
A
M
ED
PT

Fig. 2. Response surface plots illustrating the interactive effects of (a) Pumpkin puree
concentration vs Inoculum concentration, (b) Pumpkin puree concentration vs Fermentation
E

temperature, (c) Inoculum concentration vs Fermentation temperature on fermentation time

CC

(pH 4.50 set as the fermentation endpoint) of the fermented pumpkin-based beverage.
A
(a) (b)

T
R IP
(c)

SC
U
N
A
M
ED

Fig. 3. Response surface plots illustrating the interactive effects of (a) Pumpkin puree
PT

concentration vs Inoculum concentration, (b) Pumpkin puree concentration vs Fermentation

temperature, (c) Inoculum concentration vs Fermentation temperature on α-glucosidase
inhibitory activity percentage of the fermented pumpkin-based beverage after 4 weeks
E

refrigerated (4 °C) storage.

CC
A
(a) (b)

T
R IP
(c)

SC
U
N
A
M
ED
PT

Fig. 4. Response surface plots illustrating the interactive effects of (a) Pumpkin puree
concentration vs Inoculum concentration, (b) Pumpkin puree concentration vs Fermentation
E

temperature, (c) Inoculum concentration vs Fermentation temperature on L. mali survival in

the gastrointestinal condition of the fermented pumpkin-based beverage after 4 weeks
CC

refrigerated (4 °C) storage.

A
(a) (b)

T
R IP
(c)

SC
U
N
A
M
ED
PT

Fig. 5. Response surface plots illustrating the interactive effects of (a) Pumpkin puree
concentration vs Inoculum concentration, (b) Pumpkin puree concentration vs Fermentation
E

temperature, (c) Inoculum concentration vs Fermentation temperature on sensory overall

acceptability score of the fermented pumpkin-based beverage after 4 weeks refrigerated (4 °C)
CC

storage.
A
 T
R IP
SC
U
N
A
Fig. 6. Changes in α-glucosidase inhibition (%) in fermented pumpkin-based beverages
M

(obtained from optimized fermentation condition) during refrigerated (4 °C) storage in

comparison with acarbose (positive control) and non-inoculated pumpkin puree (negative
control). Each value in the table represents the mean ± SD (n = 3). Error bars represent the
ED

standard deviation of all trials. Different superscripts have mean ranks that are significantly
different from each other. Mean values with different uppercase alphabets (A-G) indicate
significantly difference (p < 0.05) among storage period (weeks); mean values with different
lowercase alphabets (a-g) indicate significant difference (p < 0.05) among samples.
E PT
CC
A
 T
R IP
SC
U
N
Fig. 7. Survival rate (%) of L. mali in the fermented pumpkin-based beverage (obtained from
A
optimized fermentation condition) during refrigerated (4 °C) storage. Error bars represent the
standard deviation of all trials. Different superscripts have mean ranks that are significantly
M

different from each other. Mean values with different lowercase alphabets (a-g) indicate
significant difference (p < 0.05) between storage period (weeks).
ED
E PT
CC
A
 T
R IP
SC
U
N
A
M
ED

Fig. 8. Seven-point hedonic sensory evaluation of the fermented pumpkin-based beverage

(obtained from optimized fermentation condition) stored at 4 °C for 4 weeks.
E PT
CC
A
Table 1
Box-Behnken design of the observed responses (fermentation time (TpH4.5), L. mali survival
in gastrointestinal condition (SGI), α-glucosidase inhibitory activity percentage (α-G) and
sensorial acceptability score (SOA) of the fermented pumpkin-based beverage) under
different experimental condition

After 4 weeks refrigerated (4 °C)

X2: storage
X1: X3:
Inoculum TpH4.5
Run Pumpkin Temperature
(Log10CFU/ (h)
(% w/v) (°C) SOA
mL) α-G (%) SGI (%)

T
(score)

IP
1 30.00 (0) 7.00 (0) 30.00 (0) 45.00 55.34 67.23 5.52

R
2 30.00 (0) 7.00 (0) 30.00 (0) 30.00 55.12 65.09 5.55

SC
3 20.00 (‒1) 6.00 (‒1) 30.00 (0) 65.00 25.01 18.04 1.89

4 30.00 (0) 6.00 (‒1) 40.00 (+1) 18.00 63.34 68.21 3.56

5 30.00 (0) 6.00 (‒1) 20.00 (‒1) 72.00

U 34.66 33.20 4.48
N
6 20.00 (‒1) 8.00 (+1) 30.00 (0) 40.00 45.42 43.33 5.40
A
7 40.00 (+1) 6.00 (‒1) 30.00 (0) 38.00 43.09 48.02 5.02
M

8 30.00 (0) 8.00 (+1) 20.00 (‒1) 63.00 72.01 70.69 4.91
ED

9 30.00 (0) 7.00 (0) 30.00 (0) 45.00 50.97 66.33 5.06

10 30.00 (0) 8.00 (+1) 40.00 (+1) 12.00 86.79 87.21 6.99
PT

11 20.00 (‒1) 7.00 (0) 20.00 (‒1) 82.00 10.12 34.46 3.30

12 30.00 (0) 7.00 (0) 30.00 (0) 44.00 55.00 60.57 5.00
E

13 30.00 (0) 7.00 (0) 30.00 (0) 40.00 55.01 67.09 5.43
CC

14 40.00 (+1) 7.00 (0) 40.00 (+1) 24.00 68.21 90.86 6.02
A

15 40.00 (+1) 8.00 (+1) 30.00 (0) 44.00 95.97 79.44 6.42

16 20.00 (‒1) 7.00 (0) 40.00 (+1) 75.00 20.01 35.37 1.96

17 40.00 (+1) 7.00 (0) 20.00 (‒1) 120.00 34.03 40.20 3.35
Table 2
Estimated coefficients of the fitted second-order polynomial models for the responses
(fermentation time (TpH4.5), L. mali survival in gastrointestinal condition (SGI), α-glucosidase
inhibitory activity percentage (α-G) and sensorial acceptability score (SOA) of the fermented
pumpkin-based beverage)

Regression coefficients estimated

Term After 4 weeks refrigerated (4 °C) storage

T
TpH4.5 (h)
α-G (%) SGI (%) SOA (score)

IP
Intercept
β0

R
40.80 54.29 65.26 5.31
Linear

SC
β1 ‒4.50*** 17.59* 15.91* 1.03*
β2 ‒4.25*** 16.76* 14.15* 1.10*
β3 ‒26.00* 10.94*
U 12.89* 0.31***
N
Interaction
β1 β 2 1.53n.s. ‒0.53**
A
7.75*** 8.12*
β1 β 3 ‒22.25* 6.07** 12.44* 1.00*
M

β2 β 3 0.75n.s. ‒3.48*** ‒4.62** 0.75**

Quadratic
ED

β11 19.98* ‒16.51* ‒16.33* ‒0.98*

β22 ‒14.03* 14.60* ‒1.72n.s. 0.35***
PT

β33 ‒14.48* ‒4.68** 1.29n.s. ‒0.68**

R2 0.98 0.99 0.99 0.99
E

CV% 10.00 4.36 3.80 5.19

Adq. precision 27.75 49.18 43.47 27.85
CC

Plof-value 0.94 0.25 0.94 0.61

Pm-value < 0.0001 < 0.0001 < 0.0001 < 0.0001
A

Subscripts: 1 = pumpkin puree concentration (20-40% w/v); 2 = inoculum concentration (6-8

Log10CFU/mL); 3 = fermentation temperature (20-40 °C).
CV%, coefficient variation [%]; Adq. Precision, adequate precision; Plof-value, probability of
F value for the lack of fit; Pm-value, probability of F value for the model.
n.s.
non-significant at p > 0.05.
*p < 0.001, **p < 0.01, ***p < 0.05.
Table 3
Estimated predicted and observed values for the responses (fermentation time (TpH4.5), L. mali
survival in gastrointestinal condition (SGI), α-glucosidase inhibitory activity percentage (α-G)
and sensorial acceptability score (SOA) of the fermented pumpkin-based beverage) within
their intervals

Response

T
After 4 weeks refrigerated (4 °C) storage
TpH4.5 (h)

IP
α-G (%) SGI (%) SOA (score)
Predicted mean 28.06 95.97 88.29 6.99

R
95% PI lowa 12.31 88.99 81.47 6.23

SC
Observed valueb 28.34 ± 0.10 95.89 ± 0.30 88.56 ± 0.67 6.99 ± 0.40
95% PI higha 43.82 102.95 95.12 7.75
a b

U
Prediction interval; Values are averages of triplicate analysis ± standard deviation of three
N
individual runs.
A
M
ED
E PT
CC
A