# Evaluation of Fructooligosaccharides and Inulins  as Potentially Health Benefiting Food Ingredients by HPAEC-PED and MALDI-TOF MS

Authors: Chiara Borromei; Maria Careri; Antonella Cavazza; Claudio Corradini; Lisa Elviri; Alessandro Mangia; Cristiana Merusi
Journal: International Journal of Analytical Chemistry
 (2009)

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## Abstract

This paper describes the complementarity of high-performance anion exchange chromatography coupled with pulsed electrochemical detection (HPAEC-PED) and matrix-assisted laser desorption/ionization mass spectrometry (MALDI-TOF-MS) to evaluate commercial available fructans (fructooligosaccharides (FOS) and inulins),  having different degrees of polymerization (DP) which are usually employed by food industry as functional ingredients either for their prebiotic properties or as a fat replacer, giving a fat-like mouth feel and texture. The developed HPAEC-PED methods are able to analyze FOS (fructans with DP 3–10) and inulins (DP ranging from 3 to 80) with a good resolution and relatively short retention times to evaluate structural differences between fructooligosaccharide and inulins and the possible presence of inulooligosaccharides as well as of branching. To characterize FOS and inulin at different degrees of polymerization and to assure correct molecular assignment, MALDI-TOF MS analysis was also investigated. The 2,5-dihydroxy benzoic acid (2,5-DHB) was found to be the best matrix for FOS analysis as Actilight and Raftilose P95 products, while 3-aminoquinoline (3-AQ) seems to be the best matrix for inulin with higher DP. The applicability of the optimized methods to the identification and determination of FOS contained in a symbiotic milk as well as a type of inulin added as functional ingredient to a cooked ham is demonstrated.


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## Body

## 1. Introduction

Fructans are carbohydrate polymers consisting of a sucrose
molecule that is elongated by a chain of fructosyl units connected throughβ-(2→1) or β-(2→6) linkages [1], depending on the linkage type
they are called inulin and levans, respectively.Inulin has been defined as a polydisperse carbohydrate
material consisting mainly, if not exclusively, ofβ-(2→1) fructosyl-fructose linkes, containing one
terminal glucose as in sucrose and having the generic chemical structure GFn
(with G as glucose, F as fructose, and n indicating DP). When referring to the definition of inulin, both GFn
and Fn compounds, consisting exclusively, of
β-(2→1) fructosyl-fructose linkes, are considered
to be included under this same nomenclature. Several inulin types occur in nature and they differ for the degree of polymerization and
molecular weight, depending on the source, the harvest time and processing
conditions [2].Fructooligosaccharides (FOS) with DP 3–9 (average DP
4.5) are produced during the process of chemical degradation or controlled
enzymatic hydrolysis of inulin by endoglycosidases [3, 4]. Furthermore,
FOS can be produced on a commercial scale, from sucrose, using a fungal enzyme
from either Aureobasidium sp. [5] or Aspergillus niger [6].FOS and inulin are recognized as health-promoting food
ingredients. The variety of
chemical and structural conformations that characterize FOS and inulins makes them flexible
and appealing ingredients for different food applications. Inulin has been reported to develop
a gel-like structure when thoroughly mixed with water or other aqueous liquid,
forming a gel with a white creamy appearance, which can be easily incorporated
into foods to replace fats making inulin an interesting ingredient to deliver
structure in low or zero fat-food products [7]. Correlation between inulin gel properties and its chemical structure (oligo- and polysaccharides) has been evaluated [8]. Furthermore, FOS and inulin exhibit prebiotic function stimulating the growth and/or activity of one or a limited number of bacteria in
the colon that can improve host health [9]
and, therefore, they can be employed in
functional food formulations [10].
In a previous work, prebiotic effectiveness of FOS and inulin of different
degrees of polymerization was reported [11], and the response of
bifidobacteria to differently lengthened fructans was analyzed in pure and fecal
cultures, confirming that fermentation of FOS and inulins in the colon can be
correlated to different metabolic activities carried out by several intestinal
microorganisms [12]. Carbohydrate analyses conducted at the end of batch
fermentations by high-performance anion-exchange
chromatography (HPAEC) with pulsed electrochemical detection (PED) technique demonstrated a very heterogeneous
strain-dependent capability to degrade FOS or inulins. It was demonstrated that
during batch fermentations, the short fructans were fermented first then
gradually the longer ones were consumed. However, regarding the investigated
carbohydrates, only qualitative indications were given with respect to the chain polymerization
degree.Within the panel of analytical techniques available for the
characterization of fructooligosaccharides (FOS) and inulin, HPAEC-PED can be a useful and sensitive tool for the
qualitative chain-length analysis of oligo- and polysaccharides from polydisperse preparations
such as FOS and inulin at different degrees of polymerization [8–11]. Besides, HPAEC-PED is routinely used to
separate neutral and charged oligosaccharides differing by branch, linkage, and
positional isomerism
[13]; from the chromatograms generated by
HPAEC it is not possible to identify each observed component, without access to
reference material.The lack of standards is an
obvious problem when investigating FOS and inulin contain linear homologous
series of fructan polysaccharides, of which there are no commercial standards
available.Matrix-assisted laser
desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) has been
used in molecular sizing of carbohydrates and the technique can conveniently be
combined with other methods such as HPLC, demonstrating to be a powerful tool
for the characterization of carbohydrates [14–16]. Furthermore,
MALDI-TOF-MS has been used to determine chain length distribution of FOS and
inulin [17, 18] as well as for both qualitative and quantitative analyses in
selected food samples [19, 20].This paper deals with the development
of HPAEC-PED methods to characterize and compare FOS and inulins having
different degrees of polymerization. The qualitative HPAEC-PED profiles were
then compared with molecular weight distribution evaluated by MALDI-TOF-MS. Our
results could be important for labeling or supporting a prebiotic claim
in food as they give indications regarding DP distribution and the amount of
FOS or inulin contained. Furthermore, this work
describes the validation and application of HPAEC-PED methods for the quantitative
determination of short-chain FOS (sc-FOS) added to a symbiotic milk and inulin
added to a cooked ham as a functional ingredient.


## 2. Materials and Methods

### 2.1. Chemicals

The deionized water (18 MΩ cm
resistivity) was obtained from a Milli-Q element water purification system
(Millipore, Bedford, Mass,
USA). Acetonitrile, methanol (both of HPLC purity), trifluoroacetic acid,
and formic acid (analytical reagent grade) were purchased from Carlo Erba
(Milan, Italy). Sodium hydroxide and
sodium nitrate were from J. T. Baker (Deventer, The Netherlands). Carrez reagent I (potassium hexacyanoferrate(II)
trihydrate), carrez reagent II (zinc acetate), glucose, fructose, and sucrose
and lactose were purchased from Sigma-Aldrich (Milan, Italy). 1-kestose was from Fluka (Milan, Italy). All sample solutions
were filtered through a Type 0.45 μm single-use membrane filter
(Millipore, Bedford, Mass,
USA).


### 2.2. Hpaec-Ped Apparatus

All HPAEC-PED experiments were performed using
a DX 500 system equipped with a GP40 pump, using a CarboPac PA-200 column (Dionex,3×250mm) and a CarboPac PA-100 (Dionex, 4×250mm), connected to the associated guard column. Carbohydrates were detected by a model ED40 electrochemical detector in its
integrated pulsed amperometric detection mode, applying the following potentials and durations: E1=0.10V(t1=0.40second), E2=−2.00V(t2=0.01second), E3=0.60V(t3=0.01second), E4=−0.10V(t4=0.06second). Integration is between 0.20 and 0.40 seconds. The chromatographic system was interfaced,
via proprietary network chromatographic software (PeakNet TM)
to a personal computer, for instrumentation control, data acquisition, and
processing. All were
from Dionex Corporation (Sunnyvale, Calif, USA).


### 2.3. Chromatographic Conditions

To separate FOS and
inulin at different degrees
of polymerization (DP), on a CarboPac PA-200 column, the
mobile phase consisted of deionized water (eluent A), 600 mM aqueous sodium
hydroxide (eluent B), and 250 mM aqueous sodium nitrate solution (eluent C),
employing a gradient program as reported in method
1, Table1. A similar procedure was developed to characterize the
oligosaccharide distribution present in a commercial available product
containing short chain fructooligosaccharides (scFOS) (Actilight
950P), in which carbohydrates were separated
on the CarboPac PA100 column, eluting by the gradient reported in method 2, Table 1, in which eluent A was water, eluent B 600 mM aqueous sodium hydroxide solution, and
eluent C 500 mM aqueous sodium acetate solution. The gradient elution program
reported in Table 2 was applied to elute oligosaccharide fraction present in
inulin added to cooked ham.Table 1
Gradient elution program to elute
(method 1) FOS and inulin and (method 2) scFOS.
HPAEC-PED method 1HPAEC-PED method 2Elution time (min)A(%)B(%)C1(%)CommentElution time (min)A(%)B(%)C2(%)Comment−40a01000Start cleaning step−45a01000Start cleaning step−30a01000End cleaning step−30a01000End cleaning step−29.9a89101Start conditioning step−29.9a89101Start conditioning step089101End conditioning step089101End conditioning step0.189101Injection, acquisition start0.189101Injection, acquisition start484151End first gradient step589101End isocratic elution45602020End first gradient step80791540End third gradient step55502030End second gradient stepA: deionized water; B: sodium hydroxide (600 mM);C1: sodium
nitrate (250 mM); C2: sodium
acetate (500 mM).Negativea time indicates time prior to sample
injection.Table 2
Gradient elution program toelute oligosaccharide
fraction present in inulin added to cooked ham.
Elution time (min)A(%)B(%)C1(%)Comment−35a01000Start cleaning step−25a01000End cleaning step−24.9a79165Start conditioning step079165End conditioning step0.1741610Injection, acquisition start20621622End first gradient stepA: deionized water; B: sodium hydroxide (600 mM); C: sodium acetate
(500 mM).Negativea time
indicates time prior to sample injection.All mobile phases were
sparged and pressurized with helium to prevent adsorption of atmospheric carbon
dioxide and subsequent production of carbonate, which would act as displacing
ion and shorten retention time.


### 2.4. Maldi-Tof-Ms Analysis of Fos

MALDI-MS measurements were performed using an MALDI-LR
time-of-flight mass spectrometer (Micromass, Manchester, UK) operating in the
positive linear ion mode. Ions formed by a pulsed UV laser beam (λ=337nm) were accelerated at 15 keV. Laser
strength was varied from sample to sample to obtain the best signal.For all samples, different matrices were tested:
2,5-dihydroxy benzoic acid (2,5-DHB) (Sigma-Aldrich), trihydroxyacetophenone
(THAP), 3-aminoquinoline (3-AQ), hydroxylphenylazo benzoic acid (HABA), and
4-hydroxy-α-alpha cyanocinnamic
acid (HCCA) (Fluka), at 10 mg mL−1 in either aqueous solution and
water/acetonitrile (50/50 v/v) trifluoroacetic acid (TFA) (0.1% v/v) mixture. A
dried droplet sample preparation was adopted. Three replicated measurements
were performed on each sample. External calibration was performed using the
[M+H]+ ions of a peptide mixture (angiotensin I, angiotensin II,
substance P, rennin, ACTH, insulin bovine, cytochrome c) (Sigma-Aldrich).


### 2.5. Samples

Different sources of fructans were
analyzed both by HPAEC-PED and MALDI-TOF-MS: Raftiline ST, Raftilose P95 (Orafti,
Tienen, Belgium), Actilight 950P (Beghin Mijie, Thumeries, France), Frutafit IQ,
and Frutafit TEX (Sensus, Roosedaal, The Netherlands). All stock solutions were
prepared at 1 mgmL−1 with HPLC-grade water and filtered on a 0.45 μm membrane filter. Cooked ham was
kindly provided by I Fratelli Emiliani SpA (Langhirano, Parma, Italy).
Symbiotic milk was purchased from the local market.


### 2.6. Sample Preparation

Symbiotic milk was analyzed for the separation of
individual sugars and short-chain fructooligosaccharides (scFOS). Two
milliliters of milk were transferred to a 25 mL volumetric flask. The sample
was diluted in approximately 10 mL ethanol-water (1:1, v/v)
and 300 μL Carrez I solution (stirred 1 minutr) and 300 μL Carrez II solution
(stirred 1 minute) were added at room temperature. Five milliliters of
acetonitrile (HPLC-grade) were added. These reagents were used to precipitate
the protein and noncarbohydrate fractions. The solution was made up to 25 mL with ethanol-water (1:1, v/v),
then the solution was left for two hours until complete formation and
precipitation of protein clot. The resulting solution diluted (1:1, v/v) with
water, then was filtered through a filter paper and passed through a C18 Sep-Pak Plus cartridge Waters (Milford, Mass, USA) previously conditioned with
10 mL of methanol (HPLC-grade) and 10 mL of HPLC grade water. This filtered
extract was forced through a 0.45 μm nylon filter and then injected into the
HPLC system.Cooked ham samples were prepared by blending two slices
(2.0 cm in width) of each ham and homogenization. Ten grams of the homogenized
sample were weighed and diluted with 50 mL of HPLC grade water and stirred with
a magnetic stirrer. The beaker with the sample was placed in a shaking
water-bath at 80°C for 60 minutes to denature proteins. The sample was
centrifuged at 7000 xg for 45 minutes at 4°C. The clarified solution was
removed and an aliquot (1 mL) was diluted with 12 mL of HPLC-grade water (final
dilution 1:60). After filtration through a 0.45 μm membrane filter, sample was
injected into HPLC.


## 2.1. Chemicals

The deionized water (18 MΩ cm
resistivity) was obtained from a Milli-Q element water purification system
(Millipore, Bedford, Mass,
USA). Acetonitrile, methanol (both of HPLC purity), trifluoroacetic acid,
and formic acid (analytical reagent grade) were purchased from Carlo Erba
(Milan, Italy). Sodium hydroxide and
sodium nitrate were from J. T. Baker (Deventer, The Netherlands). Carrez reagent I (potassium hexacyanoferrate(II)
trihydrate), carrez reagent II (zinc acetate), glucose, fructose, and sucrose
and lactose were purchased from Sigma-Aldrich (Milan, Italy). 1-kestose was from Fluka (Milan, Italy). All sample solutions
were filtered through a Type 0.45 μm single-use membrane filter
(Millipore, Bedford, Mass,
USA).


## 2.2. Hpaec-Ped Apparatus

All HPAEC-PED experiments were performed using
a DX 500 system equipped with a GP40 pump, using a CarboPac PA-200 column (Dionex,3×250mm) and a CarboPac PA-100 (Dionex, 4×250mm), connected to the associated guard column. Carbohydrates were detected by a model ED40 electrochemical detector in its
integrated pulsed amperometric detection mode, applying the following potentials and durations: E1=0.10V(t1=0.40second), E2=−2.00V(t2=0.01second), E3=0.60V(t3=0.01second), E4=−0.10V(t4=0.06second). Integration is between 0.20 and 0.40 seconds. The chromatographic system was interfaced,
via proprietary network chromatographic software (PeakNet TM)
to a personal computer, for instrumentation control, data acquisition, and
processing. All were
from Dionex Corporation (Sunnyvale, Calif, USA).


## 2.3. Chromatographic Conditions

To separate FOS and
inulin at different degrees
of polymerization (DP), on a CarboPac PA-200 column, the
mobile phase consisted of deionized water (eluent A), 600 mM aqueous sodium
hydroxide (eluent B), and 250 mM aqueous sodium nitrate solution (eluent C),
employing a gradient program as reported in method
1, Table1. A similar procedure was developed to characterize the
oligosaccharide distribution present in a commercial available product
containing short chain fructooligosaccharides (scFOS) (Actilight
950P), in which carbohydrates were separated
on the CarboPac PA100 column, eluting by the gradient reported in method 2, Table 1, in which eluent A was water, eluent B 600 mM aqueous sodium hydroxide solution, and
eluent C 500 mM aqueous sodium acetate solution. The gradient elution program
reported in Table 2 was applied to elute oligosaccharide fraction present in
inulin added to cooked ham.Table 1
Gradient elution program to elute
(method 1) FOS and inulin and (method 2) scFOS.
HPAEC-PED method 1HPAEC-PED method 2Elution time (min)A(%)B(%)C1(%)CommentElution time (min)A(%)B(%)C2(%)Comment−40a01000Start cleaning step−45a01000Start cleaning step−30a01000End cleaning step−30a01000End cleaning step−29.9a89101Start conditioning step−29.9a89101Start conditioning step089101End conditioning step089101End conditioning step0.189101Injection, acquisition start0.189101Injection, acquisition start484151End first gradient step589101End isocratic elution45602020End first gradient step80791540End third gradient step55502030End second gradient stepA: deionized water; B: sodium hydroxide (600 mM);C1: sodium
nitrate (250 mM); C2: sodium
acetate (500 mM).Negativea time indicates time prior to sample
injection.Table 2
Gradient elution program toelute oligosaccharide
fraction present in inulin added to cooked ham.
Elution time (min)A(%)B(%)C1(%)Comment−35a01000Start cleaning step−25a01000End cleaning step−24.9a79165Start conditioning step079165End conditioning step0.1741610Injection, acquisition start20621622End first gradient stepA: deionized water; B: sodium hydroxide (600 mM); C: sodium acetate
(500 mM).Negativea time
indicates time prior to sample injection.All mobile phases were
sparged and pressurized with helium to prevent adsorption of atmospheric carbon
dioxide and subsequent production of carbonate, which would act as displacing
ion and shorten retention time.


## 2.4. Maldi-Tof-Ms Analysis of Fos

MALDI-MS measurements were performed using an MALDI-LR
time-of-flight mass spectrometer (Micromass, Manchester, UK) operating in the
positive linear ion mode. Ions formed by a pulsed UV laser beam (λ=337nm) were accelerated at 15 keV. Laser
strength was varied from sample to sample to obtain the best signal.For all samples, different matrices were tested:
2,5-dihydroxy benzoic acid (2,5-DHB) (Sigma-Aldrich), trihydroxyacetophenone
(THAP), 3-aminoquinoline (3-AQ), hydroxylphenylazo benzoic acid (HABA), and
4-hydroxy-α-alpha cyanocinnamic
acid (HCCA) (Fluka), at 10 mg mL−1 in either aqueous solution and
water/acetonitrile (50/50 v/v) trifluoroacetic acid (TFA) (0.1% v/v) mixture. A
dried droplet sample preparation was adopted. Three replicated measurements
were performed on each sample. External calibration was performed using the
[M+H]+ ions of a peptide mixture (angiotensin I, angiotensin II,
substance P, rennin, ACTH, insulin bovine, cytochrome c) (Sigma-Aldrich).


## 2.5. Samples

Different sources of fructans were
analyzed both by HPAEC-PED and MALDI-TOF-MS: Raftiline ST, Raftilose P95 (Orafti,
Tienen, Belgium), Actilight 950P (Beghin Mijie, Thumeries, France), Frutafit IQ,
and Frutafit TEX (Sensus, Roosedaal, The Netherlands). All stock solutions were
prepared at 1 mgmL−1 with HPLC-grade water and filtered on a 0.45 μm membrane filter. Cooked ham was
kindly provided by I Fratelli Emiliani SpA (Langhirano, Parma, Italy).
Symbiotic milk was purchased from the local market.


## 2.6. Sample Preparation

Symbiotic milk was analyzed for the separation of
individual sugars and short-chain fructooligosaccharides (scFOS). Two
milliliters of milk were transferred to a 25 mL volumetric flask. The sample
was diluted in approximately 10 mL ethanol-water (1:1, v/v)
and 300 μL Carrez I solution (stirred 1 minutr) and 300 μL Carrez II solution
(stirred 1 minute) were added at room temperature. Five milliliters of
acetonitrile (HPLC-grade) were added. These reagents were used to precipitate
the protein and noncarbohydrate fractions. The solution was made up to 25 mL with ethanol-water (1:1, v/v),
then the solution was left for two hours until complete formation and
precipitation of protein clot. The resulting solution diluted (1:1, v/v) with
water, then was filtered through a filter paper and passed through a C18 Sep-Pak Plus cartridge Waters (Milford, Mass, USA) previously conditioned with
10 mL of methanol (HPLC-grade) and 10 mL of HPLC grade water. This filtered
extract was forced through a 0.45 μm nylon filter and then injected into the
HPLC system.Cooked ham samples were prepared by blending two slices
(2.0 cm in width) of each ham and homogenization. Ten grams of the homogenized
sample were weighed and diluted with 50 mL of HPLC grade water and stirred with
a magnetic stirrer. The beaker with the sample was placed in a shaking
water-bath at 80°C for 60 minutes to denature proteins. The sample was
centrifuged at 7000 xg for 45 minutes at 4°C. The clarified solution was
removed and an aliquot (1 mL) was diluted with 12 mL of HPLC-grade water (final
dilution 1:60). After filtration through a 0.45 μm membrane filter, sample was
injected into HPLC.


## 3. Results and Discussion

In the first part of our
work, the average degree of polymerization (DP) and distribution of oligo- and
polysaccharides in commercial FOS and inulins having different molecular weight distribution were qualitatively evaluated
by HPAEC-PED.To develop an accurate, valid, and optimal chromatographic
fingerprint for the quality evaluation of FOS and inulin at different degrees of polymerization
(DP), the different HPAEC parameters including mobile phase composition, chromatographic
column (CarboPac PA100 and CarboPac PA200), and flow rate of mobile phase were
all examined and compared. The criterion used to evaluate the quality of a
fingerprint was the number of peaks detected.Various eluent
combinations were tested using nitrate as pushing agent to enable the selective
elution with high reproducible retention time of FOS and inulin with DP up to
80 or more. Gradient elution was advantageous for separating both oligo and
polymers with different DP. Under gradient conditions, acetate ion is typically
the preferred “pusher” ion used by HPAEC. Using nitrate instead of acetate as
the pushing agent in gradient elution of carbohydrate by HPAEC, better
resolution of polymers can be achieved [21]. In our work, the dual advantages of a nitrate gradient over elution
using acetate ions were the simultaneous increase of the column peak capacity
and the reduction of the analysis time. Employing a CarboPac PA200 column,
under the chromatographic conditions described in method 1, Table 1, both low molecular
weight and high molecular weight fructans were separated (see the
chromatographic profiles reported in Figure 1). The assignment of the
chromatographic peaks with DP higher than 3 was based on the generally accepted
assumptions that the retention time of a homologous series of carbohydrates
increased as the DP increased, and that each successive peak represented a
glucofructan which had a fructose more than that of the previous peak. This is
because retention time increases as the number of negatively charged functional
groups concurrently increases [13]. Moreover, the individual peaks were sharp
and well resolved, strongly suggesting that all the analyzed samples of FOS and
fructans were, as expected, mainly linear.Figure 1
(a)
Chromatographic
profiles of standard solutions of Raftilose, (b) Frutafit TEX, (c) Frutafit IQ, (d)
Raftiline. Chromatographic conditions as in the text.Instrumental precision
was checked from six consecutive injections of an inulin solution; the relative standard
deviations (RSDs) obtained were better than 2.7%, as reported in Table3.Table 3
Repeatability of retention time (sample Frutafit
IQ).
N.PeaksRetention time±SDcv(%)N.PeaksRetention time±SDcv(%)11.90±0.040.924448.09±0.320.4021.97±0.571.524549.01±0.410.3332.10±0.092.504649.18±0.380.4842.60±0.922.064750.09±0.400.4653.12±1.522.724850.28±0.330.4764.70±2.502.474951.11±0.480.4575.73±2.062.035051.30±0.460.3589.11±2.721.945152.14±0.470.33912.38±2.471.755252.36±0.450.301015.15±2.031.915353.10±0.350.301116.38±1.942.455453.34±0.330.321218.82±1.752.555554.03±0.300.321320.36±1.912.595654.28±0.300.251422.64±2.452.625754.96±0.320.231524.52±2.552.405855.24±0.320.211626.40±2.591.655955.83±0.250.101727.00±2.621.026056.09±0.230.141828.50±2.401.126156.65±0.210.041929.92±1.650.956256.92±0.100.412031.26±1.020.806357.49±0.140.522131.55±1.120.726457.75±0.040.622232.37±0.950.636558.30±0.410.512333.79±0.800.626658.65±0.520.452434.31±0.720.576759.22±0.620.392535.64±0.630.536859.63±0.510.352635.99±0.620.516960.19±0.450.332737.33±0.570.487060.55±0.390.292837.57±0.530.267161.10±0.350.042938.89±0.510.357261.51±0.330.243039.05±0.480.317361.96±0.290.283140.26±0.260.317462.42±0.040.203240.43±0.350.257562.76±0.240.153341.62±0.310.197663.07±0.280.293441.76±0.310.517763.49±0.200.273543.04±0.250.567863.88±0.150.363643.21±0.190.317964.13±0.290.433744.21±0.510.518064.44±0.270.473844.37±0.560.358164.75±0.360.483945.52±0.310.588265.03±0.400.454045.78±0.510.398365.33±0.470.434146.72±0.350.328465.64±0.480.354247.01±0.580.418565.94±0.450.374347.87±0.390.388666.20±0.430.35Analyzing
the chromatographic profiles depicted in Figure1, the simpler chromatographic
profile was observed in sample A, which corresponds to Raftilose, where only
oligosaccharides from DP 3 to DP 9 were found.This commercially FOS product
consists of a powder composed of oligosaccharide fraction including the
trisaccharide 1-kestose and short-chain FOS at higher degree of polymerization
as well as the natural sugars glucose, fructose, and sucrose. In
chromatographic profiles depicted in Figure1, peaks
with retention times longer than 30 minutes could be assigned to polysaccharides
from DP 10 to DP 80, whereas the small peaks eluting among them could
correspond to isomers composed only of fructose unit chains [20], as well as
slightly branched fructans [21].Chromatographic profile of Frutafit
TEX (Figure1(b)) shows that most peaks were eluted with retention times
between 32 to 80 minutes, revealing that it was mainly composed by polymeric
fructans having DP higher than ten.On
the other hand,
chromatographic profile depicted in Figure1(c) was comparable to that of Figure
1(d), where a degree of polymerization number (DP) of about 80 was found for
both products.From the chromatograms generated by
HPAEC, it was not possible to identify each observed component; however, by a
qualitative comparison of the chromatographic profiles reported in Figure1, it
may be observed that the DP distribution of carbohydrates is very different.
Frutafit IQ and Raftiline were found to be characterized by a polydisperse
distribution of carbohydrates composed of both linear oligofructose (very
similar to that of Raftilose, Figure 1(a)) and polifructose at higher DP, which
correspond, regarding retention times, to that of Frutafit TEX.On
comparative grounds, it is evident from the separation profiles that HPAEC–PED allowed the
study of a higher number of oligomers, and it is able to compare fructans with different degrees of polymerization. Furthermore, pulsed amperometric detection is highly selective and
sensitive because only reactive compounds will give response and at very low concentrations.To
verify the chain length distribution of the analyzed FOS and inulin by
HPAEC-PED, the same products were analyzed by MALDI-TOF MS.As it is well known, the matrix plays a fundamental role in
quality of the MALDI-TOF MS results both in terms signal-to-noise ratio and
resolution. Before comparing
the MALDI-TOF MS profile of the FOS investigated by LC, the effects of
different matrices were tested. Various matrices, such as 2,5-DHB, 3-AQ, HCCA, and
THAP, have been previously recommended for the analysis of carbohydrates. We
started by testing these four different matrices.After several experiments, DHB was found to be the best
matrix for FOS analysis of Raftilose P95 (Figure2), while 3-AQ seems to be the
best matrix for inulin with a higher DP like Frutafit IQ (Figure 3) in which the spectrum was comparable with that obtained
analyzing Raftiline inulin sample (data not shown).Figure 2
MALDI MS spectrum of a standard solution (x mgmL−1)
of Raftilose. DHB was used as matrix. “a.i.” means arbitrary intensity and
“m/z” means the mass-to-charge ratio. Other conditions as reported in Section 2.4.Figure 3
MALDI MS spectrum of a standard solution (1.0 mgmL−1)
of Frutafit IQ. 3-AQ was used as matrix. Other conditions as reported in Figure 2.As reported in Figure4, for a inulin mainly composed of
fructans at high DP, such as Frutafit
TEX, the best matrix resulted to be THAP with fast evaporation technique.Figure 4
MALDI MS spectrum of a standard solution (1.0 mgmL−1)
of Frutafit TEX. THAP was used as matrix. Other conditions as reported in Figure 2.The MALDI-TOF mass spectra exhibited the sodium and
potassium adducts and ascribed the degree of polymerization of these fructans
(Table4). The oligomers showed the mass difference of 162 Da, which corresponds to hexose
residues and, as expected, all spectra exhibited the monomodal mass
distributions without any fragmentation [18].Table 4
Example of sodium adduct and degree of polymerization
of Raftiline standard.
Degree of polymerization (DP)[M+Na]+Degree of polymerization (DP)[M+Na]+DP3527DP172799.3DP4689DP182961.6DP5852.1DP193124DP61014.6DP203286.1DP71176.9DP213448.5DP81339.4DP223610.1DP91501.7DP233772.7DP101664DP243934.4DP111826.3DP254096.4DP121988.5DP264258.4DP132150.9DP274420.9DP142313.1DP284582.7DP152475.3DP294743.7DP162637.4DP304905.3MALDI-TOF spectra elicited the same differences of DP and
geometrical profiles between fructans, which were seen with HPAEC-PED.In MALDI-TOF spectra, there are not only visibles Raftiline
and Frutafit TEX Gaussian profiles like in chromatogram but also differences
between Raftilose maximum relative intensity (in the ranges between DP 4–6) and Frutafit TEX
maximum relative intensity (in the ranges between DP 12–14).Many papers in literature cited the use of MALDI-TOF to
analyze fructans present in natural sources as Jerusalem artichoke, onion,
shallots, elephant garlic, and so forth [17–20]. MALDI-TOF MS
gives better results for this type of food rather than HPAEC-PAD because fructans
profiles are unknown and so MALDI-TOF MS identifications need less time to optimize analysis and assure correct molecular assignment.Furthermore, MALDI-TOF MS is far less prone to contaminant
influence and does not require a tedious purification of the analytes (which
may cause selective losses of some compounds of the mixture to be analyzed)
[8].The limit of
MALDI-TOF MS is the fact that similar mass branched and linear isomers cannot
be distinguished. On the other hand, although HPAEC-PED does not allow for
structure elucidation, it permits identification of unknown carbohydrates
relative to standards whose retention behavior versus structure has been already established. A representative
elution pattern of Actilight
950P, obtained by HPAEC-PED under the chromatographic conditions reported in
the experimental part, is depicted in Figure5. Actilight, which is industrially
produced through fructosyl-transfer from sucrose using a fungal enzyme, is a
commercial available food ingredient, having the following composition of dry
substance: 0.3% fructose, 0.4% glucose, 3.0% sucrose, 36% 1-kestose (GF2), 49%
nystose (GF3), and 12% fructosyl-nystose (GF4) [22]. From the chromatographic
profile A depicted in Figure 5, it is possible to clearly identify the free
monosaccharide glucose and fructose, the unreacted disaccharide sucrose, and
the trisaccharide 1-kestose, which were identified eluting the corresponding
standards. Other oligosaccharides were selectively eluted, but the
identification of the individual oligosaccharides was a challenging task due to
lack of suitable standards. However, the retention times of carbohydrates from
the HPAEC column depend both on DP and structural differences (e.g., branching)
and evaluating the chromatographic profiles of Figure 5, we can concluded that peak no. 6 and peak no. 7
can be assigned as the tetrasaccharide nystose (GF3) and the pentasaccharide 1F-fructosyfuranosyl-nystose
(GF4), respectively.Figure 5
Chromatographic profiles of (a) symbiotic milk containing
Actilight as prebiotic scFOS; (b) synthetic mixture of glucose, fructose,
sucrose, lactose, and 1-kestose (GF3); (c) Actilight. Chromatographic conditions as reported in the text. Peak
identification: (1) glucose, (2) fructose, (3) sucrose, (4) lactose, (5) 1-kestose,
(6) GF4, (7) GF5.Moreover, the chromatographic profile of the commercial
scFOS preparation shows at least other four unknown oligomers that should be
assumed as inulooligosaccharides ranging from F2 to F5 [23].The unit chain length distribution of scFOS in the
Actilight
product was analyzed by MALDI-TOF MS, and the
obtained spectrum confirmed that the analyzed product was a mixture of short-chain
oligosaccharides ranging from DP 3 to DP 5, having a mass difference of 162 Da
(results not shown). Although, a distinction
of oligomers having similar masses branched and linear isomers is not possible
by MALDI—TOF—MS, the use of this
mass spectrometric technique was
a very useful tool for the molecular weight measurement, whereas the heterogeneity of scFOS distribution in Actilight was confirmed through the chromatographic
profile obtained by HPAEC-PED.


### 3.1. Food Applications

#### 3.1.1. Symbiotic Milk

According to a widely accepted definition, a functional
food is any modified food that has special effects on the human organism beyond
the nutrients it contains [24].Milk with added functional
ingredients, but not fermented, is proposed in the market of functional foods. In
these functional dairy, products are included unfermented milk with added probiotic
and prebiotics, called symbiotic, due to their symbiotic functional action.A representative
pattern of a carbohydrate elution profile of commercial symbiotic functional
milk is depicted by the chromatographic profile B of Figure5. The main peaks
in the chromatogram obtained on the CarboPac PA 100 column, using the elution
program reported in method 2, Table 1, consist of glucose, sucrose, lactose,
and scFOS. Besides the identified compounds, as reported in the chromatographic
profile A, the chromatographic profile B shows the presence of minor peaks which can be
identified as previously reported. Chromatographic profile C is referred to a
standard solution of glucose, fructose, sucrose, lactose, and 1-kestose. The validation process of the
optimized HPLC-PED method was carried out
following the EURACHEM guidelines [25].Table5
summarizes the precision of retention times observed upon injecting the same
sample of milk and using the optimized experimental conditions. The
instrumental precision was evaluated by repeating the analysis of the same milk
sample six times. As can be seen, relative standard deviations (RSDs) of
retention times were lower than 2.10% (n=6) for the same day, while
these values increase up to 2.55% when the same experiment was repeated in six
different days (n=24). Furthermore, method repeatability was evaluated
using the same data obtained for the accuracy study, where the RSDs of peak
areas were in all experiments better than 2.85%.Table 5
Reproducibility of retention times for intra- and interday analyses for the symbiotic milk sample by HPAEC-PED (separation conditions as in Figure5).
Intraday (n=6)Interday (6 days,n=24)CompoundRetention
time
(min)RSD
(%)Retention
time
(min)RSD
(%)Glucose5.751.885.932.24Fructose6.872.087.052.75Saccharose8.901.9110.102.60Lactose12.411.8212.622.441-kestose15.901.7716.102.10Quantification was based on external
standard method. The assay linearity was determined by the analysis of
six different concentrations of the standard solutions. Each level of concentration was prepared in
triplicate. The linearity of response for the analyzed sugars was demonstrated
at six different concentrations from 50 to 300μg/mL for glucose,
fructose, sucrose, and 1-kestose and from 50 to 450 μg/mL for lactose,
respectively. The standard curves were obtained by plotting peak area (y)
versus nominal concentration x (μgmL−1) of each compound and were
fitted to the linear regression. The standard deviation (SD) of slope and
intercept was estimated at the 95% confidence level.The limit of
detection (LOD) was defined as three times the standard deviation of the blank
values (Sb) divided by the slope of the calibration curves,
whereas the limit of quantification (LOQ) was defined as 10 Sb divided by the slope of the calibration curve. LOD for all analyzed samples was
ranging from 10.0 to 12.5 μg/mL and LOQ
from 25.0 to 32.0 μg/mL,
respectively.Quantification of scFOS was determined from peak area using
1-kestose as the external standard. As commercial GF4 and GF5 standards
were not available, they were not quantified and Actilight was quantified
considering that 1-kestose represents
the 36% of the whole product. The recoveries, measured at three
concentration levels, varied from 97.0 to 104.3%. The validated method was
successfully applied to the simultaneously determination of glucose, fructose,
sucrose, lactose, and scFOS in symbiotic milk, obtaining the following results:
glucose 0.58 (±0.08) mg/mL, sucrose 0.62 (±0.07) mg/mL, lactose 44.76 (±0.52) mg/mL, 1-kestose
7.28 (±0.11) mg/mL. Nystose (GF3) 78.7 mg/mL, fructofuranosylnistose (GF4) 56.8 mg/mL, and GF5 12.5 mg/mL. From the above results and data reported in literature, the
content of scFOS in the examined symbiotic milk can be evaluated to be within
2.0 (±0.2)% (w/v). Furthermore, no variation of peak area ratio of 1-kestose-GF3 and
1-kestose-GF4 was noticed
over ten days, demonstrating that scFOS composition did not change over the whole shelf life of
the product.


#### 3.1.2. Cooked Ham

Inulin was added to the brine used in the industrial
process for preparation of commercial cooked ham, prior the cooking step, as a
replacer of sugars with the aim of reducing caloric content. The inulin profile
of ham was analyzed by the HPAEC PED method described in method 1, Table1. Comparing chromatographic profiles reported in Figure 6,
there were no observable differences in the inulin profiles between those
extracted from (b) the cooked ham and (a) the control, corresponding to inulin
which was added to the brine solution. Furthermore, the same chromatographic
profiles were also observed during the whole shelf life of the product (data not shown).Figure 6
Chromatographic profiles of a standard solution (0.5 mgmL−1) of (a) Frutafit IQ compared with (b) the chromatographic profile of an
extract of inulin from a cooked ham sample. Chromatographic conditions as in the text.To perform quantitative evaluation of inulin present
in cooked ham, we selected six unidentified peaks of the oligosaccharide fraction, which were selectively eluted
within 20 minutes using the gradient elution program reported in Table2 (see
Figure 7). To verify the quality and usefulness of the method, the
analytical parameters linearity, sensitivity, precision, and percentage of
recovery were determined. The linearity of response for the selected
unidentified peaks was demonstrated at six different concentrations of inulin,
ranging from 50 to 300 μg/mL.
Higher concentrations
were not assayed because we considered that the range was wide enough for the
proposed applications. The linearity of the present method for all unidentified
oligosaccharides selected as reference peaks was good, with correlation
coefficients higher than 0.997. The limit of detection was evaluated for each
of the eluted peaks,
and results are
summarized in Table 6. The recoveries, measured at three concentration levels
of inulin added to the ham sample after homogenization,
varied from 91 to 106%.Table 6
Limits of detection and quantitation.
Peaks123456Limit of detection(yD)aμg/g12.2113.6512.4513.2214.2716.03Limit of quantification(yQ)bμg/g43.3248.6249.8940.6750.7751.21a Concentration corresponding to signal and yD=yb+2t(95%, n−1)sb.b Concentration corresponding to signal and yQ=yb+10sb.Figure 7
Chromatographic profile of an extract of inulin from a cooked ham sample,
showing inside the circle the oligosaccharide fraction selected to perform quantitative evaluation of inulin present
in the analyzed sample. Chromatographic conditions as in the text.Result
regarding quantitative determination of inulin in cooked ham gave results
ranging from3.20±0.20 to 3.00±0.42, dry matter and on the average
1.05 (±0.02)g on 100 g of cooked ham.


## 3.1. Food Applications

### 3.1.1. Symbiotic Milk

According to a widely accepted definition, a functional
food is any modified food that has special effects on the human organism beyond
the nutrients it contains [24].Milk with added functional
ingredients, but not fermented, is proposed in the market of functional foods. In
these functional dairy, products are included unfermented milk with added probiotic
and prebiotics, called symbiotic, due to their symbiotic functional action.A representative
pattern of a carbohydrate elution profile of commercial symbiotic functional
milk is depicted by the chromatographic profile B of Figure5. The main peaks
in the chromatogram obtained on the CarboPac PA 100 column, using the elution
program reported in method 2, Table 1, consist of glucose, sucrose, lactose,
and scFOS. Besides the identified compounds, as reported in the chromatographic
profile A, the chromatographic profile B shows the presence of minor peaks which can be
identified as previously reported. Chromatographic profile C is referred to a
standard solution of glucose, fructose, sucrose, lactose, and 1-kestose. The validation process of the
optimized HPLC-PED method was carried out
following the EURACHEM guidelines [25].Table5
summarizes the precision of retention times observed upon injecting the same
sample of milk and using the optimized experimental conditions. The
instrumental precision was evaluated by repeating the analysis of the same milk
sample six times. As can be seen, relative standard deviations (RSDs) of
retention times were lower than 2.10% (n=6) for the same day, while
these values increase up to 2.55% when the same experiment was repeated in six
different days (n=24). Furthermore, method repeatability was evaluated
using the same data obtained for the accuracy study, where the RSDs of peak
areas were in all experiments better than 2.85%.Table 5
Reproducibility of retention times for intra- and interday analyses for the symbiotic milk sample by HPAEC-PED (separation conditions as in Figure5).
Intraday (n=6)Interday (6 days,n=24)CompoundRetention
time
(min)RSD
(%)Retention
time
(min)RSD
(%)Glucose5.751.885.932.24Fructose6.872.087.052.75Saccharose8.901.9110.102.60Lactose12.411.8212.622.441-kestose15.901.7716.102.10Quantification was based on external
standard method. The assay linearity was determined by the analysis of
six different concentrations of the standard solutions. Each level of concentration was prepared in
triplicate. The linearity of response for the analyzed sugars was demonstrated
at six different concentrations from 50 to 300μg/mL for glucose,
fructose, sucrose, and 1-kestose and from 50 to 450 μg/mL for lactose,
respectively. The standard curves were obtained by plotting peak area (y)
versus nominal concentration x (μgmL−1) of each compound and were
fitted to the linear regression. The standard deviation (SD) of slope and
intercept was estimated at the 95% confidence level.The limit of
detection (LOD) was defined as three times the standard deviation of the blank
values (Sb) divided by the slope of the calibration curves,
whereas the limit of quantification (LOQ) was defined as 10 Sb divided by the slope of the calibration curve. LOD for all analyzed samples was
ranging from 10.0 to 12.5 μg/mL and LOQ
from 25.0 to 32.0 μg/mL,
respectively.Quantification of scFOS was determined from peak area using
1-kestose as the external standard. As commercial GF4 and GF5 standards
were not available, they were not quantified and Actilight was quantified
considering that 1-kestose represents
the 36% of the whole product. The recoveries, measured at three
concentration levels, varied from 97.0 to 104.3%. The validated method was
successfully applied to the simultaneously determination of glucose, fructose,
sucrose, lactose, and scFOS in symbiotic milk, obtaining the following results:
glucose 0.58 (±0.08) mg/mL, sucrose 0.62 (±0.07) mg/mL, lactose 44.76 (±0.52) mg/mL, 1-kestose
7.28 (±0.11) mg/mL. Nystose (GF3) 78.7 mg/mL, fructofuranosylnistose (GF4) 56.8 mg/mL, and GF5 12.5 mg/mL. From the above results and data reported in literature, the
content of scFOS in the examined symbiotic milk can be evaluated to be within
2.0 (±0.2)% (w/v). Furthermore, no variation of peak area ratio of 1-kestose-GF3 and
1-kestose-GF4 was noticed
over ten days, demonstrating that scFOS composition did not change over the whole shelf life of
the product.


### 3.1.2. Cooked Ham

Inulin was added to the brine used in the industrial
process for preparation of commercial cooked ham, prior the cooking step, as a
replacer of sugars with the aim of reducing caloric content. The inulin profile
of ham was analyzed by the HPAEC PED method described in method 1, Table1. Comparing chromatographic profiles reported in Figure 6,
there were no observable differences in the inulin profiles between those
extracted from (b) the cooked ham and (a) the control, corresponding to inulin
which was added to the brine solution. Furthermore, the same chromatographic
profiles were also observed during the whole shelf life of the product (data not shown).Figure 6
Chromatographic profiles of a standard solution (0.5 mgmL−1) of (a) Frutafit IQ compared with (b) the chromatographic profile of an
extract of inulin from a cooked ham sample. Chromatographic conditions as in the text.To perform quantitative evaluation of inulin present
in cooked ham, we selected six unidentified peaks of the oligosaccharide fraction, which were selectively eluted
within 20 minutes using the gradient elution program reported in Table2 (see
Figure 7). To verify the quality and usefulness of the method, the
analytical parameters linearity, sensitivity, precision, and percentage of
recovery were determined. The linearity of response for the selected
unidentified peaks was demonstrated at six different concentrations of inulin,
ranging from 50 to 300 μg/mL.
Higher concentrations
were not assayed because we considered that the range was wide enough for the
proposed applications. The linearity of the present method for all unidentified
oligosaccharides selected as reference peaks was good, with correlation
coefficients higher than 0.997. The limit of detection was evaluated for each
of the eluted peaks,
and results are
summarized in Table 6. The recoveries, measured at three concentration levels
of inulin added to the ham sample after homogenization,
varied from 91 to 106%.Table 6
Limits of detection and quantitation.
Peaks123456Limit of detection(yD)aμg/g12.2113.6512.4513.2214.2716.03Limit of quantification(yQ)bμg/g43.3248.6249.8940.6750.7751.21a Concentration corresponding to signal and yD=yb+2t(95%, n−1)sb.b Concentration corresponding to signal and yQ=yb+10sb.Figure 7
Chromatographic profile of an extract of inulin from a cooked ham sample,
showing inside the circle the oligosaccharide fraction selected to perform quantitative evaluation of inulin present
in the analyzed sample. Chromatographic conditions as in the text.Result
regarding quantitative determination of inulin in cooked ham gave results
ranging from3.20±0.20 to 3.00±0.42, dry matter and on the average
1.05 (±0.02)g on 100 g of cooked ham.


## 3.1.1. Symbiotic Milk

According to a widely accepted definition, a functional
food is any modified food that has special effects on the human organism beyond
the nutrients it contains [24].Milk with added functional
ingredients, but not fermented, is proposed in the market of functional foods. In
these functional dairy, products are included unfermented milk with added probiotic
and prebiotics, called symbiotic, due to their symbiotic functional action.A representative
pattern of a carbohydrate elution profile of commercial symbiotic functional
milk is depicted by the chromatographic profile B of Figure5. The main peaks
in the chromatogram obtained on the CarboPac PA 100 column, using the elution
program reported in method 2, Table 1, consist of glucose, sucrose, lactose,
and scFOS. Besides the identified compounds, as reported in the chromatographic
profile A, the chromatographic profile B shows the presence of minor peaks which can be
identified as previously reported. Chromatographic profile C is referred to a
standard solution of glucose, fructose, sucrose, lactose, and 1-kestose. The validation process of the
optimized HPLC-PED method was carried out
following the EURACHEM guidelines [25].Table5
summarizes the precision of retention times observed upon injecting the same
sample of milk and using the optimized experimental conditions. The
instrumental precision was evaluated by repeating the analysis of the same milk
sample six times. As can be seen, relative standard deviations (RSDs) of
retention times were lower than 2.10% (n=6) for the same day, while
these values increase up to 2.55% when the same experiment was repeated in six
different days (n=24). Furthermore, method repeatability was evaluated
using the same data obtained for the accuracy study, where the RSDs of peak
areas were in all experiments better than 2.85%.Table 5
Reproducibility of retention times for intra- and interday analyses for the symbiotic milk sample by HPAEC-PED (separation conditions as in Figure5).
Intraday (n=6)Interday (6 days,n=24)CompoundRetention
time
(min)RSD
(%)Retention
time
(min)RSD
(%)Glucose5.751.885.932.24Fructose6.872.087.052.75Saccharose8.901.9110.102.60Lactose12.411.8212.622.441-kestose15.901.7716.102.10Quantification was based on external
standard method. The assay linearity was determined by the analysis of
six different concentrations of the standard solutions. Each level of concentration was prepared in
triplicate. The linearity of response for the analyzed sugars was demonstrated
at six different concentrations from 50 to 300μg/mL for glucose,
fructose, sucrose, and 1-kestose and from 50 to 450 μg/mL for lactose,
respectively. The standard curves were obtained by plotting peak area (y)
versus nominal concentration x (μgmL−1) of each compound and were
fitted to the linear regression. The standard deviation (SD) of slope and
intercept was estimated at the 95% confidence level.The limit of
detection (LOD) was defined as three times the standard deviation of the blank
values (Sb) divided by the slope of the calibration curves,
whereas the limit of quantification (LOQ) was defined as 10 Sb divided by the slope of the calibration curve. LOD for all analyzed samples was
ranging from 10.0 to 12.5 μg/mL and LOQ
from 25.0 to 32.0 μg/mL,
respectively.Quantification of scFOS was determined from peak area using
1-kestose as the external standard. As commercial GF4 and GF5 standards
were not available, they were not quantified and Actilight was quantified
considering that 1-kestose represents
the 36% of the whole product. The recoveries, measured at three
concentration levels, varied from 97.0 to 104.3%. The validated method was
successfully applied to the simultaneously determination of glucose, fructose,
sucrose, lactose, and scFOS in symbiotic milk, obtaining the following results:
glucose 0.58 (±0.08) mg/mL, sucrose 0.62 (±0.07) mg/mL, lactose 44.76 (±0.52) mg/mL, 1-kestose
7.28 (±0.11) mg/mL. Nystose (GF3) 78.7 mg/mL, fructofuranosylnistose (GF4) 56.8 mg/mL, and GF5 12.5 mg/mL. From the above results and data reported in literature, the
content of scFOS in the examined symbiotic milk can be evaluated to be within
2.0 (±0.2)% (w/v). Furthermore, no variation of peak area ratio of 1-kestose-GF3 and
1-kestose-GF4 was noticed
over ten days, demonstrating that scFOS composition did not change over the whole shelf life of
the product.


## 3.1.2. Cooked Ham

Inulin was added to the brine used in the industrial
process for preparation of commercial cooked ham, prior the cooking step, as a
replacer of sugars with the aim of reducing caloric content. The inulin profile
of ham was analyzed by the HPAEC PED method described in method 1, Table1. Comparing chromatographic profiles reported in Figure 6,
there were no observable differences in the inulin profiles between those
extracted from (b) the cooked ham and (a) the control, corresponding to inulin
which was added to the brine solution. Furthermore, the same chromatographic
profiles were also observed during the whole shelf life of the product (data not shown).Figure 6
Chromatographic profiles of a standard solution (0.5 mgmL−1) of (a) Frutafit IQ compared with (b) the chromatographic profile of an
extract of inulin from a cooked ham sample. Chromatographic conditions as in the text.To perform quantitative evaluation of inulin present
in cooked ham, we selected six unidentified peaks of the oligosaccharide fraction, which were selectively eluted
within 20 minutes using the gradient elution program reported in Table2 (see
Figure 7). To verify the quality and usefulness of the method, the
analytical parameters linearity, sensitivity, precision, and percentage of
recovery were determined. The linearity of response for the selected
unidentified peaks was demonstrated at six different concentrations of inulin,
ranging from 50 to 300 μg/mL.
Higher concentrations
were not assayed because we considered that the range was wide enough for the
proposed applications. The linearity of the present method for all unidentified
oligosaccharides selected as reference peaks was good, with correlation
coefficients higher than 0.997. The limit of detection was evaluated for each
of the eluted peaks,
and results are
summarized in Table 6. The recoveries, measured at three concentration levels
of inulin added to the ham sample after homogenization,
varied from 91 to 106%.Table 6
Limits of detection and quantitation.
Peaks123456Limit of detection(yD)aμg/g12.2113.6512.4513.2214.2716.03Limit of quantification(yQ)bμg/g43.3248.6249.8940.6750.7751.21a Concentration corresponding to signal and yD=yb+2t(95%, n−1)sb.b Concentration corresponding to signal and yQ=yb+10sb.Figure 7
Chromatographic profile of an extract of inulin from a cooked ham sample,
showing inside the circle the oligosaccharide fraction selected to perform quantitative evaluation of inulin present
in the analyzed sample. Chromatographic conditions as in the text.Result
regarding quantitative determination of inulin in cooked ham gave results
ranging from3.20±0.20 to 3.00±0.42, dry matter and on the average
1.05 (±0.02)g on 100 g of cooked ham.


## 4. Conclusions

We have demonstrated the usefulness of MALDI-TOF MS, and that high-pH anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD) provides
powerful tools for the analysis of FOS and inulins with a high sensitivity and
no need for derivatization.HPAEC-PED methods were
developed to determine chain length distribution of FOS and inulins, which were
compared with the corresponding distributions from MALDI-TOF MS analyses.
Although some differences were observed, the developed HPAEC-PED methods can be
considered as secondary or orthogonal methods complementary to MALDI-TOF MS to
evaluate oligo- and
polysaccharides distribution in the analyzed samples. MALDI-MS gives
better assurance of correct molecular assignment since the isotopic mass of
each peak is available, although similar masses branched and linear isomers
cannot be distinguished.By using HPAEC and MALDI-TOF MS
analysis, the presence of FOS and inulin at different degrees of polymerization
could neither be demonstrated in ingredient preparations nor from
functionalized foods.



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*Source: 530639-2009-03-18.xml*