**3. Results and discussion**

#### **3.1 Spectral signature of GG and LBG**

Galactomannans are polysaccharides formed by a linear (1-4)-β-D-mannan backbone with a D-galactose side chain (**Figure 4**). On average, GG has a single α-D-galactopyranosyl unit connected by (1–6) linkages to every second main chain unit. In the case of the LBG, unsubstituted or sparingly (1–4) substituted regions of mannopyranose units and regions heavily substituted with α-D-galactopyranosyl residues attached by (1–6)-bonds have been observed.

The galactose substitution that influences the intrinsic flexibility of mannan backbone causes solubility differences and controls the rheological properties. The FTIR-ATR signatures of GG and LBG (**Figure 5**) show essentially difference in intensity due to the mannose/galactose ratio and the presence of residual chemical compounds from thermo-mechanical and chemical dehusking pretreatments (remaining germ particles, products of thermal degradation of endosperm, etc.). In GG, the endosperm is composed of 75% of galactomannose and the rest consists of pentosan, protein, pectin, phytin, ash, and dilute acid insoluble residues [5]. High protein content (like albumin,

**Figure 4.** *Molecular structure of LBG (a) and GG (b) from [14].*

**Figure 5.** *FTIR-ATR spectra of LBG and GG.*

globulin, and glutelin [2]) could be found in LBG because of a greater contamination by germ particles; lipids were also detected, just like the presence of rhamnose, arabinose, xylose, and glucose, contaminants proceeding from the seed coat [23].

The broadband between 3700 and 3000 cm<sup>−</sup><sup>1</sup> describes the O–H and N–H stretching vibration (hydrogen bonding) attributed to water, amide, and carbohydrates. The spectral zone between 3000 and 2800 cm<sup>−</sup><sup>1</sup> contains two bands assigned to C–H stretching vibration in methylene groups of rings (νas CH2 at 2925 cm<sup>−</sup><sup>1</sup> and νs CH2 at 2871 cm<sup>−</sup><sup>1</sup> ). The carbonyl signal at 1743 cm<sup>−</sup><sup>1</sup> (C〓O stretching vibration) could be in relation with the amide group of amino acids. A spectrum of GG reference material does not reveal the bands of these contaminants [10]. The band pointed at 1640 cm<sup>−</sup><sup>1</sup> is due to the presence of bound water (O–H bending of absorbed water), which cannot be eliminated despite the freeze-drying. This last band could also be attributed to the axial deformation of C〓O bond (amide band I) and the one pointed at 1527 cm<sup>−</sup><sup>1</sup> could correspond to the angular deformation of N–H bond (amide band II) or to amine N–H deformation vibrations due to the presence of impurities such as proteins and amino acids found in the germ and seed coat, badly remaining during the purification process [24].

The peaks observed in the spectra between 1480 and 1190 cm<sup>−</sup><sup>1</sup> represented C–H, C–OH (1236 cm<sup>−</sup><sup>1</sup> ), and H–C–H deformation vibration (bending). Depending on the level of protein impurities, a band at 1236 cm<sup>−</sup><sup>1</sup> could be assigned to the amide band III (C–N) vibration mode [24]. The following spectral region (1190–900 cm<sup>−</sup><sup>1</sup> ) presents different bands contributing to the skeletal vibrations and glycosidic bonds (νCC, νCOC, νCCO, and δOCH) of galactomannans' sugar composition. The C–O stretching mode of pyranose ring exists as a small shoulder at 1055 cm<sup>−</sup><sup>1</sup> and a broad peak at 1012 cm<sup>−</sup><sup>1</sup> , while the shoulder at 966 cm<sup>−</sup><sup>1</sup> is a characteristic contribution of C–OH bending [25]. At lower wavenumbers (900–700 cm<sup>−</sup><sup>1</sup> ), peaks at 806 and 870 cm<sup>−</sup><sup>1</sup> appear that are related with anomeric C–H deformation bands (CCH and OCH) of structural isomers (α or β-pyranose compounds), equatorial C–H deformation bands (nonglycosidic), and skeletal symmetric and asymmetric ring vibrations (CCO, COC, and OCO) [26, 27].

The FTIR-ATR spectrum of each sugar is presented in **Figure 6**.

These compounds have a CH2OH labile group capable of generating intra- and intermolecular hydrogen bonding. This CH2OH labile group and the arrangement of the other OH group on pyranose ring affect differently the spectral region between 3500 and 3000 cm<sup>−</sup><sup>1</sup> . The reversed hydroxyl group orientation in the C-2 and C-4 atom in the

**83**

700 cm<sup>−</sup><sup>1</sup>

**Figure 6.**

.

*Discrimination by Infrared Spectroscopy: Application to Micronized Locust Bean and Guar Gums*

structure results in differences in positions and intensities of several bands in the region

ging and twisting, C–O and C–C stretching vibration of backbone, and COH bending of primary and secondary bonded alcohol appear. Because of the equatorial and axial

differences about skeletal stretching (CO, CC, and COC) vibration coupled with (COH,

PCA, carried out on all of the normalized infrared spectra of gums, shows that on the components 1 and 2, which represent respectively 57 and 24% of the total spectral variance, the samples of GG in red and LBG in blue form two groups perfectly separated from each (**Figure 7**). The best separation between clusters was obtained with a selection of variables in anomeric region, between 1450 and

The mid-infrared technique allows characterizing the commercial samples of GG and LBG. Globally, all GG samples are negatively projected; LBG samples are positively projected along PC1, while PC2 is a representative of the intragroup variability. The spectral band characteristics of GG and LBG powders are observable on PC1 loading (**Figure 8**). The negative part of PC1 loading characterize more LBG samples by exalting

the proteins whose content appears more important in the case of LBG. The band at 1170 could be attributed to C–O vibrations of locust bean galactomannan. The large dispersion of locust bean samples along PC1 could be due to the presence of impurities in gum powders coming from husk and germ, (like insoluble matter, proteins, amino acid, etc.), residual compounds resulting from the various steps of extraction, and purification processes. Different maturity stages of seed, geographic origin, and climatic conditions could also be responsible for the variability of the chemical composition of these galactomannan gums. The positive part of PC1 reveals

OH position, the glycosidic and anomeric regions (1000–700 cm<sup>−</sup><sup>1</sup>

CCH, and CCO) deformation bands (**Table 2**) [27, 28].

spectral bands pointed at 1386, 1315, and 1236 cm<sup>−</sup><sup>1</sup>

), between 1400 and 1100 where bands of CH2 wag-

) show also spectral

(amide bands) characterizing

*DOI: http://dx.doi.org/10.5772/intechopen.87568*

of CH2 scissoring (1495–1420 cm<sup>−</sup><sup>1</sup>

*FTIR-ATR spectra of D-mannose and D-galactose.*

**3.2 PCA of gum FTIR-ATR spectra**

*Discrimination by Infrared Spectroscopy: Application to Micronized Locust Bean and Guar Gums DOI: http://dx.doi.org/10.5772/intechopen.87568*

**Figure 6.** *FTIR-ATR spectra of D-mannose and D-galactose.*

*Modern Spectroscopic Techniques and Applications*

globulin, and glutelin [2]) could be found in LBG because of a greater contamination by germ particles; lipids were also detected, just like the presence of rhamnose, arabinose, xylose, and glucose, contaminants proceeding from the seed coat [23].

stretching vibration (hydrogen bonding) attributed to water, amide, and carbohy-

to C–H stretching vibration in methylene groups of rings (νas CH2 at 2925 cm<sup>−</sup><sup>1</sup>

tion) could be in relation with the amide group of amino acids. A spectrum of GG reference material does not reveal the bands of these contaminants [10]. The

absorbed water), which cannot be eliminated despite the freeze-drying. This last band could also be attributed to the axial deformation of C〓O bond (amide band

of N–H bond (amide band II) or to amine N–H deformation vibrations due to the presence of impurities such as proteins and amino acids found in the germ and seed

to the amide band III (C–N) vibration mode [24]. The following spectral region

and glycosidic bonds (νCC, νCOC, νCCO, and δOCH) of galactomannans' sugar composition. The C–O stretching mode of pyranose ring exists as a small shoul-

is a characteristic contribution of C–OH bending [25]. At lower wavenumbers

C–H deformation bands (CCH and OCH) of structural isomers (α or β-pyranose compounds), equatorial C–H deformation bands (nonglycosidic), and skeletal symmetric and asymmetric ring vibrations (CCO, COC, and OCO) [26, 27]. The FTIR-ATR spectrum of each sugar is presented in **Figure 6**.

These compounds have a CH2OH labile group capable of generating intra- and intermolecular hydrogen bonding. This CH2OH labile group and the arrangement of the other OH group on pyranose ring affect differently the spectral region between 3500

), and H–C–H deformation vibration (bending).

) presents different bands contributing to the skeletal vibrations

. The reversed hydroxyl group orientation in the C-2 and C-4 atom in the

). The carbonyl signal at 1743 cm<sup>−</sup><sup>1</sup>

describes the O–H and N–H

is due to the presence of bound water (O–H bending of

could correspond to the angular deformation

, while the shoulder at 966 cm<sup>−</sup><sup>1</sup>

appear that are related with anomeric

contains two bands assigned

represented

could be assigned

(C〓O stretching vibra-

and

The broadband between 3700 and 3000 cm<sup>−</sup><sup>1</sup>

νs CH2 at 2871 cm<sup>−</sup><sup>1</sup>

**Figure 5.**

band pointed at 1640 cm<sup>−</sup><sup>1</sup>

*FTIR-ATR spectra of LBG and GG.*

C–H, C–OH (1236 cm<sup>−</sup><sup>1</sup>

(1190–900 cm<sup>−</sup><sup>1</sup>

der at 1055 cm<sup>−</sup><sup>1</sup>

(900–700 cm<sup>−</sup><sup>1</sup>

and 3000 cm<sup>−</sup><sup>1</sup>

I) and the one pointed at 1527 cm<sup>−</sup><sup>1</sup>

drates. The spectral zone between 3000 and 2800 cm<sup>−</sup><sup>1</sup>

coat, badly remaining during the purification process [24].

The peaks observed in the spectra between 1480 and 1190 cm<sup>−</sup><sup>1</sup>

Depending on the level of protein impurities, a band at 1236 cm<sup>−</sup><sup>1</sup>

and a broad peak at 1012 cm<sup>−</sup><sup>1</sup>

), peaks at 806 and 870 cm<sup>−</sup><sup>1</sup>

**82**

structure results in differences in positions and intensities of several bands in the region of CH2 scissoring (1495–1420 cm<sup>−</sup><sup>1</sup> ), between 1400 and 1100 where bands of CH2 wagging and twisting, C–O and C–C stretching vibration of backbone, and COH bending of primary and secondary bonded alcohol appear. Because of the equatorial and axial OH position, the glycosidic and anomeric regions (1000–700 cm<sup>−</sup><sup>1</sup> ) show also spectral differences about skeletal stretching (CO, CC, and COC) vibration coupled with (COH, CCH, and CCO) deformation bands (**Table 2**) [27, 28].

## **3.2 PCA of gum FTIR-ATR spectra**

PCA, carried out on all of the normalized infrared spectra of gums, shows that on the components 1 and 2, which represent respectively 57 and 24% of the total spectral variance, the samples of GG in red and LBG in blue form two groups perfectly separated from each (**Figure 7**). The best separation between clusters was obtained with a selection of variables in anomeric region, between 1450 and 700 cm<sup>−</sup><sup>1</sup> .

The mid-infrared technique allows characterizing the commercial samples of GG and LBG. Globally, all GG samples are negatively projected; LBG samples are positively projected along PC1, while PC2 is a representative of the intragroup variability. The spectral band characteristics of GG and LBG powders are observable on PC1 loading (**Figure 8**).

The negative part of PC1 loading characterize more LBG samples by exalting spectral bands pointed at 1386, 1315, and 1236 cm<sup>−</sup><sup>1</sup> (amide bands) characterizing the proteins whose content appears more important in the case of LBG. The band at 1170 could be attributed to C–O vibrations of locust bean galactomannan. The large dispersion of locust bean samples along PC1 could be due to the presence of impurities in gum powders coming from husk and germ, (like insoluble matter, proteins, amino acid, etc.), residual compounds resulting from the various steps of extraction, and purification processes. Different maturity stages of seed, geographic origin, and climatic conditions could also be responsible for the variability of the chemical composition of these galactomannan gums. The positive part of PC1 reveals


#### **Table 2.**

*Spectral interpretations of pure sugars.*

#### **Figure 7.**

*Representation of GG and LBG samples in (PC1 and PC2) plan (normalized spectral data).*

the most intense spectral bands in GG samples at 1066, 1012, 964, 863, 819, and 771 cm<sup>−</sup><sup>1</sup> . Bands at 1012 and 964 cm<sup>−</sup><sup>1</sup> are attributed to C–O–H and C–O vibrations, respectively. The band at 771 cm<sup>−</sup><sup>1</sup> is due to ring stretching and ring deformation of β-D-(1-4) and α-D-(1-6) linkages. These last ones are specific to the anomeric region where C–O stretching bands are more representative because of the largest number of galactose units (1-6) linked to β-D-mannopyranosyl backbone in GG.

While GG was richer in galactosyl residue, no specific bands of D-galactose were found in positive part of PC1 loading representing the GG samples. The comparison with the spectral bands of pure D-galactose or D-mannose is not a good way because of their crystalline structure (free form) that is not the case in their polymer form. Another explanation could be a possible interference with the presence of water that modifies the band's resolution in the anomeric region as it is observable in **Figure 9** presenting sugar profiles under crystalline and hydrated forms.

**85**

**Figure 9.**

**Figure 8.** *PC1 loading.*

**3.3 Prediction of species origin by PLS-1-DA regression**

The classification into varietal origin (GG or LBG) was performed using PLS-1-DA analysis. The calibration dataset was composed of 37 GG and 13 LBG (n = 50 samples × 3 spectra = 150). Different samples of calibration set, 12 LBG and 37 GG (n = 49 samples × 3 spectra = 147), have constituted the validation set.

*Spectral profiles of D-mannose (a) and D-galactose (b) under crystalline and hydrated forms.*

*Discrimination by Infrared Spectroscopy: Application to Micronized Locust Bean and Guar Gums*

*DOI: http://dx.doi.org/10.5772/intechopen.87568*

*Discrimination by Infrared Spectroscopy: Application to Micronized Locust Bean and Guar Gums DOI: http://dx.doi.org/10.5772/intechopen.87568*

**Figure 9.** *Spectral profiles of D-mannose (a) and D-galactose (b) under crystalline and hydrated forms.*
