potentiometric biosensor for determination of … · in this study, a simple and selective...

92

ISSN 2348 – 0319 International Journal of Innovative and Applied Research (2017)

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Volume 5, Issue 9

Journal home page: http://www.journalijiar.com

RESEARCH ARTICLE

POTENTIOMETRIC BIOSENSOR FOR DETERMINATION OF LACTOSE USING KEFIR GRAINS

MODIFIED ON MULTI-WALLED CARBON NANOTUBES/CASEIN COMPOSITE.

*Mohammad Mahdi Doroodmand and Farideh Zare.

Department of Chemistry, College of Sciences, Shiraz University, 71454, Shiraz, Iran.

*Corresponding Author:- Mohammad Mahdi Doroodmand.

……………………………………………………………………………………………………… Abstract:

In this study, a simple and selective biosensor has been introduced for determination of lactose by

potentiometric method based on the catalytic behavior of kefir grains via modification with multi-walled carbon

nanotubes (MWCNTs)/casein composite as indicator electrode and sat’d Ag/AgCl as reference electrode. To

fabricate the indicator electrode, kefir grains were initially dried using a freeze dryer during 48 h time interval and

powdered with an electronic grinder. Then ~ 0.15 g kefir powder was physically mixed with MWCNTs/casein with

optimized weight ratio of 0.005/0.3 (w/w) and mixed with ~ 150 µL nujol oil. The generated paste was then packed

inside a Teflon tube (i.d: 10 mm, height: 100 mm) and adopted as indicator electrode. Maximum potentiometric

response was also observed at pH 7.0 and 0.51 M ionic strength, controlled using phosphate buffer (0.01 M) and

NaCl (0.5 M), respectively. The linear dynamic range during lactose determination was ranged between 1.0×10-12

-

1.0×10-4

M with detection limit of 3.0×10-14

M based on extrapolation definition. Small hysteresis (lower than ~4%)

was observed during sequential analyses of several lactose solutions with different molar concentrations. The

response time based on the 90% of maximum response (t90) was estimated to be ~2 min. The validity of this method

was evaluated via direct determination of lactose in some types of milk samples. This biosensor was applicable for

indirect lactose determination via estimation of the end point of lactose-containing milk sample during titration by a

dilute HCl solution (1.0 mM) as titrant.

Key Words:- Kefir grains; Lactose; MWCNTs/Casein composite biosensor.

………………………………………………………………………………………………………

Introduction:- The dairy industry has made several attempts to improve the quality and the nutrient contents of milk-based

formula. In order to develop special products for specific people, some ingredients have been added to the milk

powder [1]. For example, calcium is added to prevent osteoporosis that frequently affects old people, while some

vitamins and minerals are used in the formulae for the pregnant women [1]. Recently enriched milk with

carbohydrates and carbohydrate-reduced production has been existed.

Lactose is the main carbohydrate in the dairy products and is considered as the only saccharide synthesized

by mammals at a concentration of 4–5% (w/v) in cow and other mammals, and ∼8% in the human milk [1-5]. This

disaccharide is catabolized into the glucose and galactose monosaccharides by the lactase enzyme [1, 5-7]. Lactose

has many important physiological functions. For instance, it takes part in the metabolism of calcium to help the

absorption of calcium in the human body [8]. It also plays an important role in the formation of the neural system

and the growth of skin (texture), bone skeleton and cartilage in infants. So it can prevent rickets and saprodontia [1,

8]. This compound is able to develop the producing of bacillus in babies’ bowels too [8].

Each year, daily productions suffer from great economic losses due to cow mastitis [5]. It is a serious

disease in dairy animals causing reduction in milk yield as well as lowering its nutritive values such as lactose level

[5, 9]. Lactose in the dairy products is also necessary for the cheese industry in order to control the fermentation

processes [5]. Lactose-controlled products are especially important in lactose-reduced or lactose-free products,

because ~75% of adults worldwide suffer from lactose intolerance (LI) [2, 5, 10]. Lactose intolerance or lactose

maldigestion simply describes the incomplete digestion and subsequent malabsorption of this reagent [2, 11].

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Determination of lactose has been considered by scientists during the last years. Various methods have

been used for lactose determination such as gravimetry [12], colorimetry [13], high-performance liquid

chromatography [1], and infrared spectroscopy [14]. In spite of introduction of various methods for determination of

lactose, the common points often in all the reviewed articles are that, these analytical techniques are sometimes

limited due to problems such as difficulties, low selectivity, narrow linearity, high cost, toxic reagent, low sensitivity

and/or less improved detection limits [12-14]. All of these limitations reveal strong demand for selective, sensitive

and fast determination of lactose in the presence of other carbohydrates in different real samples. For this purpose in

this research a new method is introduced using kefir grains, for electrochemical determination of lactose during the

catalytic process.

Briefly, kefir has been originally made in the "Balkans, Eastern Europe and the Caucasus" [15-17]. It

differs from other fermented products in its starter, which exists in the form of grains, while in most cases yoghurt

and cheese that contain some bacteria are used as starter cultures [18, 19]. The grains are elastic, slimy, and varying

from white to light yellow in color, which are generally ranged between 1-3 cm in length [19, 20]. They behave as

biologically vital organisms and grow, propagate and pass their properties on to the following generations of new

grains.

The microflora of the kefir grains is remarkably stable. This compound also retains its activity for years, if

preserve and incubate under appropriate cultural and physiological conditions [18]. Kefir grains contain a complex

microbial symbiotic mixture of lactic acid bacteria (LAB), acetic acid bacteria, lactose/non-lactose -fermenting

yeasts, and mycelia-forming fungi [17, 20-23]. This cluster of microorganisms is held together by a polysaccharide,

which is named as “Kefiran”, and protein matrix [17, 21, 23].

Material and Methods:- Materials:-

All the chemical reagents were from their analytical grades. To fabricate the electrode system multi-walled

carbon nanotubes (MWCNTs) with ~99% purity percentage were synthesized by chemical vapor deposition (CVD)

method according to the scanning electron microscopic image (SEM) shown in Fig. 1. For this purpose, acetylene

(Pasbaloon, Shiraz, Iran) was selected as the source of carbon in an inert atmosphere of Ar/He (1:1, V/V, Linde,

Germany) at 1200 oC inside a quartz tubing situated in a tubing furnace. Also ~ 15.0 weight percentage solution was

prepared using ferrocene (Merck) in toluene (Fluka, analytical grate) and around 1% (V/V) thiophene (Fluka) for

lengthening the CVD-synthesized MWCNTs. Kefir grains were purchased from market (Shiraz, Iran, home-made,

Purchased date: March, 2014). Lactose.H2O solution (Fluka, GMW: 360.32 g mol-1

) was prepared via dissolving

0.901 g lactose in a 250 mL-volumetric flask. The ionic strength of the solution was set to 0.5 M using NaCl

solution (Merck).

To fabricate the kefir/MWCNT composite, silica grease (Labtron Co.) was selected as nujol oil

(LABTRON CO., density: 1.32 g mL-1

). The pH of the solution was set to 7.0 using 0.01 M phosphate buffer

(H2PO4-/HPO4

2-, Merck). Analytical grade of He gas with 99.93 % purity percentage was adopted to separate any

dissolved oxygen (DO) from the solution before each potentiometric analysis. The interfering effects of foreign

species such as Ca2+

, Mg2+

, K+, Fe

3+, Cu

2+, Zn

2+, Cl

-, CH3COO

-, PO4

3- (all of them were from Merck Companies) as

well as some organic species such as D (+)-glucose.H2O (Merck), D (+)-sucrose (Fluka), Glycine (Flucka), Adonit

(Merck), Vitamins C and B6 (from Darou Pakhsh-Iran Company) were evaluated via individually dissolving at least

100-fold excess of the species in 10.0 μM lactose standard solution. Some milk suspensions and milk powder

samples (Kalleh, Damdaran, bebelac Company) were selected as the real samples during evaluation of the reliability

of the recommended procedure and method. Analytical grades of SiO2 (Merck), melamine formaldehyde (Fars

chemical industry) and casein (Sigma Aldrich) were also selected during construction of the kefir-based electrode.

In addition, hydroxyl apatite nanoparticles were synthesized according to the procedure reported in Ref. [25].

Universal (0.01 M) and boric acid solution (1.0 M) (H3BO3 crystals, Merck) were used for conditioning the indicator

electrode. 50.0-mL standard solutions (0.01 M) were prepared individually via dissolving 99.1, 37.5, 76.1, and 171.2

mg glucose, glycine, adonit, and sucrose, respectively.

Apparatus:-

A centrifuge (model: Hettich) with 5000 rpm was used for elimination of interfering agents. A pH meter

(Model: Metrohm 827) was utilized to control the pH of the prepared solution. A potentiometer (model: Lutron pH-

208) was adopted for the potentiometric system using a two-electrode system including the kefir/MWCNT

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composite as indictor electrode and Ag/AgCl (Sat'd Cl-) as reference electrode at 25

oC inside a Faraday's cage. A

DO meter (model: Lutron DO-5510) was used to measure the DO during experiment.

Fabrication of kefir/MWCNTs composite:-

To fabricate the indicator electrode, kefir grains were initially dried using a freeze drier (model: Christ

ALPHA 2-4 LV pluse) during 48 h time interval at -87 oC temperature and ~1.0 mbar pressure. Afterward they were

grinded using an electronic grinder to change them to powder. Then ~ 0.15 g kefir powder was physically mixed

with MWCNTs/casein with optimized weight ratio of 0.017 (w/w) using 2.5 mg MWCNTs and 0.15 g casein and

mixed with about 150 µL the nujol oil. 30, 0.5, 30, and 39.5 % (w/w) of kefir powder, MWCNTs, casein, and nujol

oil, respectively, were used. The generated paste was then packed inside a Teflon tube (i.d: 10 mm, height: 100 mm)

and adopted as indicator electrode and then conditioned inside borate buffer (pH ~9) for ~ 20 h.

Procedure:-

To determine the amount of lactose in milk, briefly, the solution was initially centrifuged for 10 min for

elimination of fats and fat-soluble Vitamins. Then the pH of the each milk sample was enhanced to ~11 using NaOH

(Sat'd) to generate probable metal hydroxyl precipitation as well as formation of crystal of casein-Vitamin. This

precipitation was then separated from the milk serums by centrifuging at 5000 rpm during 10 min time interval.

Then the pH of the solution was fixed at the isoelectric pH of casein (i.e. pH~ 6.4) by addition of HCl solution. This

process led to separate the excess quantity of casein inside the milk solution during the centrifuging [25]. After that

the milk sample was diluted 10 times. Then 1.46 g NaCl was added into 5.0 mL diluted milk sample and the solution

was transferred to a 50.0-mL volumetric flask and diluted with phosphate buffer (pH~7, 0.01 M). Because of the

effect of ionic strength on the activity of H+, the pH was decreased to some extent; therefore it was set to 7.0 by

saturated NaOH solution. After that the DO was removed from the solution via purging He gas (purity: 99.9996 %)

with flow rate of several mL min-1

for ~2 min. Finally the potentiometric response of the solution was measured

using the two-electrode system (Indicator and Ag/AgCl (Sat’d Cl- electrodes) during ~ 2 min as the response time of

the electrode system. At this condition, the shelf time of the fabricated sensor was estimated to around 30 days.

Real sample analysis:-

To evaluate the quantity of lactose in the milk samples, the electrode system was initially conditioned for

~1.0 min inside the prepared milk sample solution (serum) and then the potentiometric responses of the solutions

were measured. The sensitivity (p-[Lactose]/decade) of the biosensor was estimated via calculation of the slope of

the calibration curve. Then the concentration of lactose in each real sample (50.0 mL) was estimated via difference

between the potentiometric response (ΔE) of the unknown solution ([lactose]u) before and after spiking a standard

lactose concentration (Molarity: 1.0 mM, volume: 8.0 mL, [lactose]s, Eq. 1). The reliability of these results was also

evaluated via comparison between the results obtained from the experiment and the titrimetry based on the

procedure reported in Ref. [12] as reference analytical method for lactose determination (Eq. 1).

Results and discussion:- In this study for the first time the analytical properties of the kefir grains have been considered as catalytic

species for fabrication of a novel type of biosensor for lactose determination by potentiometry. Parameters having

strong influence during determination of lactose include: size of kefir grains, optimum ratio of

kefir/MWCNTs/casein, quantity of nujol oil, effect of pH and ionic strength of the electrolyte solution, and de-

aeration of the electrolyte solution. The parameters were optimized by one-at-a time method.

Kefir grains:-

One of the most important factors during formation of homogeneous composite as electrode was the size of

the kefir grains. Kefir matrix due to the possessing alive micro-organisms such as bacteria and yeast tends to form

colonies, resulting in the formation of kefir grains. The size and morphology of kefir grains were strongly dependent

to several factors such as the percentage and concentration of fats inside the initial milk, which acted as suitable

nutrient (mother solution) during the growth of kefir grains. The kefir grains were inserted into milk (%Fat=2.5) at

dark condition for 2-3 days. At these conditions, kefir grains were grown, followed by separation from the mother

solution simply via decanting the milk suspension. After washing the kefir grains with ~ 0.5 L triply-distillated

water, the kefir grains were slowly dried via heating by a blow dryer for a few minutes. Then the dried kefir grains

were introduced to the freeze dryer based on the recommended conditions. After that they were grinded using an

electronic grinder (model: Bosch) to get the kefir powder. The size and morphology of the kefir grains before and

after introduction to the freeze drying process have been graphically shown in Fig. 2. The average diameter of the

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kefir grains after growth in the nutrient medium was estimated to be between 1-2 cm, whereas these values were

reduced to ~ 0.1 mm after applying the freeze drying process. This procedure therefore led to provide kefir grains as

small as possible during formation of composite matrix with maximum homogeneity.

MWCNTs as conductive support:-

To have indicator electrode with maximum conductivity, presence of conductive species such as carbon

nanostructures was also necessary. Among different types of carbon allotropes, MWCNTs due to processing

significant characteristics such as presence of plenty of –COOH and –OH functional groups in their matrix, as well

as because of having lots of edge planes played role as excellent conductive support during formation of electrode in

the electrochemical processes.

Another important property of the MWCNTs was the biocompatibility of this carbon allotrope with micro-

organism and kefir grains [26]. However, for further confidence about the nutrient behavior of humidified

MWCNTs, the kefir gains were grown in the presence of MWCNTs inside an incubator under sterilized process in

the atmosphere of 5% CO2 in air for about one weak. The results are graphically shown in Fig. 3. Based on the

observations, MWCNTs not only were biocompatible with the kefir grains, but also acted as acceptable nutrient

medium during the growth of the kefir grains.

Effect of weight ratio of MWCNTs/kefir grains:-

Different weight ratios of MWCNTs/kefir grains were used to optimize the kefir-based composite electrode.

The results have been shown in Table 1. Based on the results, MWCNTs/kefir grain weight ratios below the 1:10

and higher than 1:60 were not suitable. This was because the share of kefir grains was negligible compared to

MWCNTs at ratios below 1:10. Whereas at ratios higher than 1:60, not only it was needed to use more kefir grains

but also no significant sensitivity was observed. These results pointed to the importance of the weight ratios of

MWCNTs/kefir on the mechanism of the electrochemical process during potentiometric determination of lactose. It

seemed that, competition between these two factors was the responsible for such behavior of the adopted electrode

system that should be evaluated during proposing the probable mechanism.

Effect of casein in the kefir-based composite:-

To have selective potentiometric sensor with fast response time, presence of a water insoluble support in the

electrode matrix was important. This process led to rapid interaction of the analyte with the surface of the indicator

electrode based on different mechanisms such as electrostatic interaction, physic/chemical adsorption, etc. [27]. For

a molecular species such as lactose as analyte, this process probably obeyed physical mechanisms such as

adsorption. For this purpose, effect of some solid supports such as melamine formaldehyde, hydroxyl apatite

nanoparticles, SiO2, and casein was evaluated in detail.

In this electrode system, weight ratios of 0.8447, 0.0141, 0.0004, and 0.1408 were selected for kefir grains,

activated MWCNTs, nujol oil, and the solid support, respectively, under similar experimental conditions. It should

be noted that, difference between the sensitivity of the electrodes during lactose analysis was related to the effect of

solid supports on the mechanism of the process.

Based on the results, maximum potentiometric linear range was observed during doping casein in the kefir

grain/MWCNT composite matrix. This effect was probably attributed to the effective role of casein during

formation of micro-emulsion with lactose. This process not only promoted the selective redox interaction of lactose

with the support, but also majorly enhanced the charge transfer process on the surface of the electrode system.

Therefore, casein as a green and biocompatible matrix was selected as the solid support during the construction of

the kefir-based composite electrode.

To optimize the matrix of the indicator electrode, different weight ratios between 0.62-0.143, 0.002-0.01,

0.33-0.378 and 0.04-0.477 were selected for kefir grains, MWCNTs, nujol oil, and casein, respectively, and the

performance of the electrode system was evaluated during analysis of a lactose standard solution with 0.1 μM

concentration under similar condition. The results were graphically shown in Fig. 4.A and summarized in Table 2.

As clearly shown, maximum sensitivity was observed during selecting weight ratios of 29.1, 0.48, 51.1,

19.36 (Electrode #3) and 0.3, 0.005, 0.395, 0.3 (Electrode #5) for the kefir grains, MWCNTs, nujol oil, and casein

composites, respectively. At these conditions, the behavior was initially attributed to various probable phenomena

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such as i) occurrence of both anodic or cathodic redox processes depending on the weight percentages of casein in

the electrode matrix, or ii) formation of various species that strongly affected the potentiometric response during

lactose determination. As reverse behaviors were observed for the response of the two tested electrodes,

consequently continuously optimization was achieved individually on the two types of this electrode system.

Effect of pH:-

As the redox process of some organic electroactive species was strongly dependent on pH, to reach the

highest sensitivity as well as to interpret the probable existing mechanism during the redox of lactose, effect of pH

was evaluated in detail. To adopt more biocompatible buffer solution, phosphate buffer was selected for controlling

the pH of the electrolyte solutions. For this purpose, phosphate solution (0.01 M) was adopted to provide solutions

with the pH values such as 4.5, 6.0, 6.5, 7.0, 9.0, and 11.0. The results have been shown in Fig. 4.B. Based on the

results (Fig. 4.B); maximum sensitivity was observed at pH ~7.0 for the two types of analyzed indicator electrodes.

This effect is also considered as a good phenomenon during analysis of biological and food samples that often have

neutral pH value. However, the same behaviors were observed for the two types of the indicator electrodes that

pointed to the effective role of pH or ionic strength on the mechanism of redox of lactose on the surface of the

introduced kefir-based composite electrode.

Effect of ionic strength:-

To identify the probable mechanism of the electrochemical process during the lactose analysis as well as to

reach the highest sensitivity for the electrochemical reaction of lactose on the surface of the electrode, effect of ionic

strength was also evaluated in detail. For this purpose, NaCl solution was selected as suitable probe. The results

related to the analysis of a lactose standard solution (0.1 μM) at different ionic strengths ranging between 0.01 – 0.5

M have been shown in Fig. 4.C. As is clearly exhibited (Fig. 4.C), the highest sensitivity was observed at ionic

strength of 0.5 M. So, this ionic strength was selected as optimum value. Therefore, both pH and ionic strength had

important roles during the potentiometric determination of lactose.

Effect of oxygen:-

Probably due to the salting-out effect of ionic species on the concentration of dissolve oxygen, effect of ionic

strength was considered as surprising note. Based on the results in spite of using phosphate buffer (0.01 M) at pH

~7.0, extra addition of NaCl showed a major effect on redox of lactose on the surface of the electrode system.

Consequently, further researches such as evaluation of the effect of DO were needed to propose the probable

mechanism during redox of lactose by the recommended method. To evaluate the effect of the DO, the

potentiometric responses were evaluated before and after purging the electrolyte solution with O2 and He with a

flow rate of around several mL min-1

for ~10 min. The results are shown in Fig. 5.A during analysis of lactose

solution (0.1 μM) at the optimum conditions.

Based on the results, reverse behavior was observed during purging He to the electrolyte solution. In

addition, more intensive response was observed during de-aeration (~10 min) of the electrolyte solution using an

inert gas such as He or N2 (Fig. 5.B). Therefore, DO in the electrolyte solution played a significant role during

analysis of lactose on the surface of kefir-based electrode system. Based on the results, salting-out effect of NaCl

probably decreased the solubility of DO in the electrolyte solution.

Effect of CO2 as the product of the redox reaction:-

To identify the probable mechanism of this biosensor, the response of the electrode system was evaluated

during analysis of a lactose standard solution before and after purging CO2 gas with a flow rate of around several

mL min-1

for ~ 5 min. No significant change was observed in the response time during lactose (1.0 mM) analysis

before and after purging CO2, but major increase in the sensitivity was observed during following the potentiometric

response. The results are shown in Fig. 6.A.

According to increasing effect of dissolved CO2 on the sensitivity of the biosensor, CO2 was considered as

one of the important products during the oxidation of lactose. To have full confidence about the formation of CO2

during the oxidation process, the concentration of dissolved CO2 was indirectly evaluated via following the

concentration of HCO3- during analysis of a concentrated lactose (1.0 mM) at a short time ( ~ 10 min) at pH ~7.0.

For this purpose acid/base titrimetry was selected using 1.0 mM HCl standard solution. However, due to the

interfering effect of phosphate buffer during the titrimetry, the pH of the solution was set to ~7.0 using a strong

acid/base such as HCl and NaOH.

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According to the titration curve (Fig. 6.B), the pH value at half point of the titration curve was estimated to

be 6.5, which was attributed to the pK1 of H2CO3/HCO3-. Therefore, CO2 was considered as the product of the

reaction during lactose analysis. The concentration of the dissolved CO2 was estimated to be ~ 5.0×10-6

M (n=2). As

this concentration was surprising and not acceptable for a potentiometric technique at zero current, therefore the

quantity of CO2 was evaluated during contacting the lactose solution to a fixed 150/2.5/150 mg of

kefir/MWCNTs/casein support and 150 μL nujol oil during purging He to the solution.

Based on the results, no CO2 was generated during de-aeration of the solution, whereas ~ 1.0×10-4

M CO2

was generated when the concentration of dissolved O2 was estimated to 6.0 parts per million detected using a ref.

DO meter. This result pointed to the effective role of O2 during decomposition of lactose and formation of CO2. In

another word, the indicator electrode sensed lactose via two independent processes including i) reaction 1 through

the catalytic effect of micro-organisms and ii) reaction 2 based on an half anodic reaction via oxidization of lactose

to CO2, situated inside the kefir matrix in the presence of DO. However due to the operation of the potentiometric

sensor at zero current (open circuit), only a negligible sense was considered for the indicator electrode through the

electrochemical process.

Effect of glucose as intermediate reagent:-

One of the characteristics of micro-organisms was their ability to hydrate lactose during formation of

glucose. To reach the formation of glucose as the intermediate of the product, potentiometric response of the kefir-

based biosensor during the analysis of a glucose standard solution (1.0 mM) was evaluated in detail. The trace

diagram (potential vs. time) was shown in Fig. 7.

Based on the results (Fig. 7), the kefir-based biosensor was sensitive to glucose. For further investigation of

the effective role of glucose, another monosaccharide such as pentose was selected as a suitable probe. Based on the

observation, no sensitive potentiometric response was observed during analysis of 1.0 mM pentose standard

solution. Therefore, among the monosaccharides, probably only glucose was considered as intermediate during the

oxidation of lactose.

Surprisingly, in this study glucose was considered as only an intermediate species that was generated during

the hydrolysis of lactose on the surface of the electrode. The lack of interfering effect of the coexisting glucose in

the milk was probably attributed to the change in its structure into its reducing form during the sample preparation

inside the dilute acidic conditions according to a report [28]. Whereas the glucose generated on the surface of the

indicator electrode was appeared in the non-reducing form. Fig. 7 also shows the trace (potential vs. time) during

potentiometric analysis of lactose standard solution (1.0 mM) spiked with glucose (1.0 mM) before and after

controlling the pH ~ 7.0. Consequently, besides lactose, kefir grains had capability for selective determination of

glucose generated by the lactose.

Probable mechanism of the lactose oxidation:-

Based on the evidences, it was concluded that, lactose was initially hydrated to glucose. After that glucose

was converted to CO2 through both the reaction 1 (in the presence of dissolve oxygen) and reaction 2 processes on

the surface of the electrode according to the following schemes (Scheme. 1 and 2).

Formation of glucose as intermediate species was also evaluated indirectly by titrimetry during estimation of

the end point of lactose-containing milk sample, titrated by a dilute HCl solution (1.0 mM) as titrant. The titration

curve has been shown in Fig. 8. Based on the results, interaction between glucose and dilute HCl led to form

reducing glucose. This result again revealed the formation of glucose as intermediate species during lactose

detection and determination. The lack of any interference of glucose probably presented inside the milk sample was

therefore evidenced via formation of reducing glucose according to the titrimetry.

Analytical figures of merit:-

The calibration curve (diagram of potential vs. log [Lactase]) was illustrated in Fig. 9.A. The introduced

method was suitable for lactose determination yielding a linear dynamic range from 1.0×10-4

to 1.0×10-12

M. The

detection limit (DL) was defined as the lactose concentration by the extrapolation. According to this definition, the

limit of detection was found as 3.0×10-14

M (Fig. 9.B).

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The sensitivity of this method was evaluated according to the slope (calibration sensitivity) of the calibration

curve (Fig. 9.A). These values were evaluated to 6.0 p [Lactose]/decade. Reproducible results were obtained during

several analyses (n= 3) of 0.1×10-5

M lactose. No significant difference (maximum hysteresis: ~4.0%, Fig. 10) was

observed in the potentiometric response of the kefir-based biosensor during several replicate analyses of 10.0 μM

lactose at different periods of time. This phenomenon clearly revealed the stability of the fabricated kefir-based

biosensor during selective determination of lactose. To evaluate the selectivity of the fabricated kefir-based

biosensor during the quantitation process, the probable interfering effect of at least 100-fold excess of various

foreign species such as lactic acid, adonit, glycine, Fe3+

, Cu2+

, Ca2+

, Mg2+

, K+, PO4

3- , Cl

-, CH3COO

-, sucrose,

aqueous vitamins on 10.0 μM lactose were checked out. Among these species, Ca

2+ had ~ 70 % interference effect

on determination of lactose. Fortunately Ca2+

was simply precipitated and removed from the sample matrix based on

the recommended procedure. Consequently this method was considered as selective method for determination of

lactose in sophisticated matrices such as milk.

Real sample analysis:-

The validation of the method was evaluated by analytical tests on some milk samples via comparison between this

method and titrimetry as reference and accepted analytical method, followed by evolution of the relative error

percentage. The results are shown in Table 3. Good agreement was obtained during comparing the results of this

method with those evaluated using titrimetry, revealing the reliability and acceptance of this method for lactose

determination. This biosensor was also applicable for indirect glucose determination during estimation of the end

point of glucose-containing milk sample, titrated by a diluted HCl solution (1.0 mM) as titrant (Fig. 8). The

stoichiometric ratio of glucose to HCl was estimated to be 1:4. Consequently this method can be applied for both

detection of lactose and glucose in the milk samples.

Conclusions:-

In this work, a sensitive and reproducible biosensor has been introduced for lactose determination based on

the catalytic behavior of kefir grains. Comparison between the proposed technique and previously reported methods

for lactose determination is shown in Table 4. As clearly shown, this technique has revealed some beneficial aspects

such as more simplicity, high selectivity, fast response time and acceptable detection limit.

Acknowledgment:- The authors wish to acknowledge the support of this work by the Shiraz University Research Council.

Fig. 1:- TEM image of MWCNTs (Purity: 99%. i.d.: 20-50, functionalized group: -COOH and –OH).

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Fig.

2:-

Image

s of

kefir

grains

A)

before

and

B), C)

after

introd

uction to the freeze dryer for 24 and 48 hours, respectively.

Fig. 3:- Optical microscopic images of kefir grains A) initial time and B) after one week growth in the presence of

MWCNTs as support, chocolate as nutrient medium and gram positive and gram negative bacteria inside incubators

at 37 oC and 5.0% CO2 in air atmosphere.

Fig. 4:- A) Effect of different solid supports on the potentiometric responses during analysis of lactose with the

optimized weight ratio of MWCNTs/kefir grains electrodes at room temperature, B) optimization of casein ratio

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with the optimized weight ratio of MWCNTs/kefir grains during analysis of 1.0×10-7

M lactose at room temperature

and C) effect of pH on the sensitivity of lactose using two indicator electrodes during analysis of 1.0×10-7

M lactose

using phosphate solution (0.01 M) at the optimized conditions (n=3).

Fig. 5:- A) effect of ionic strength on the potentiometric responses of lactose indicator electrodes during analysis of

lactose with 1.0×10-7

M concentration at pH ~7.0 at the optimized conditions (n=3) and B) effect of dissolved O2

and de-aeration electrolyte solution on the potentiometric response of lactose indicator electrode during analysis of

1.0 mM lactose at the optimized conditions.

Fig. 6:- Images including A) effect of dissolved CO2 on the sensitivity of lactose during analysis of lactose with 1.0

mM concentration at the optimized conditions (n=3) and B) titration curves during independent analysis of HCO3-

by HCl (1.0 mM) as titrant.

Fig. 7:- Potentiometric trace (diagram of potential vs. time) during analysis of a glucose standard solution (1.0 mM)

at the optimized condition (n=3).

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Fig. 8:- Titration curve of a lactose-containing milk solution titrated with HCl (1.0 mM).

Fig. 9:- A) Calibration curve for lactose determination at the optimized condition and B) the DL of lactose (M).

Fig. 10:- Hysteresis of the sensor during analyses of different concentrations of lactose.

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Scheme 1:- Proposed mechanism during potentiometric determination of lactose.

Scheme 2:- Catalytic hydration and oxidation of lactose by kefir grains.

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Table 1:- Effect of weight ratio of MWCNTs/kefir grains during analysis of lactose.

Table 2:- The optimization of casein ratio.

Table 3:- Validation of this method.

Table 4:- Comparison between present study and previously reported methods for lactose determination.

1Ratios

(MWCNTs/Kefir grains, w/w)

Equation Linear Range (M) Correlation

Coefficient (R2)

(1:10) y = 2.1377 x + 90.314 1.0×10-2

- 1.0×10-8

0.730

(1:15) y = 0.2305 x + 32.085 1.0×10-3

- 1.0×10-8

0.100

(1:25) y = 2.0758 x + 82.765 1.0×10-3

- 1.0×10-8

0.860

(1:30) y = 1.4631 x + 55.943 1.0×10-3

- 1.0×10-8

0.890

(1:40) y = 4.5955 x + 82.308 1.0×10-2

- 1.0×10-8

0.900

(1:45) y = 3.3558 x + 69.202 1.0×10-3

- 1.0×10-8

0.860

(1:60) y = 2.2498 x + 45.77 1.0×10-4

- 1.0×10-12

0.985 1The data are the average of 3 independent analyses.

1 Electrode

Number

Eblank (mv) Elactose (mv) E (mv) t90 % (min) %W/W casein

1 44.88 32.06 -12.82 0.033 12.08

2 51.09 65.01 13.92 7.2 15.42

3 14.26 82.6 68.34 9.0 19.36

4 57.2 46.72 -10.48 2.7 21.47

5 117.89 63.34 -54.55 13.5 30.00

6 36.29 38.8 2.51 2.5 38.34

7 49.81 42.53 -7.28 0.033 40.1

8 31.54 34.95 3.41 0.033 44.15

9 39.13 41.19 2.06 0.033 47.69 1 The data are the average of 3 independent analyses.

Real Samples 1Introduced

Method

(g per 100 g)

Titrimetry as Reference

Method

(g per 100 g)

Relative Error

Percentage (%)

Lactose-free Milk (Kalleh Co.) 0.50 0.52 - 3.8

Lactose-free Milk (Damdaran

Co.)

0.21 0.22 - 4.5

Lactose-free Milk powder

(Bebelac Co.)

0.58

0.56 +3.6

1 The data are the average of 3 independent analyses.

References Method Detection

Limit

(g mL-1

)

Linear Range

(g mL-1

)

References

Third generation amperometric biosensor 9.0×10-8

1.7×10-7

–

6.85×10-5

[29]

Enzyme reactor with co-immobilization of β-galactosidase

and glucose oxidase

2.7×10−8

8 × 10−8

– 4 ×

10−6

[30]

Lactose biosensor by microelectrodes pre-modified with

Pt/graphene/P

1.3×10-6

5.0×10-6

–

6.0×10-5

[31]

Potentiometric biosensor for determination of lactose

using kefir grains

1.0×10-14

3.4×10-13

–

3.4×10-5

Present

study

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potentiometric biosensor for determination of … · in this study, a simple and selective...

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