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Influence of germination time on the morphological,

morphometric, structural, and physicochemical characteristics of
Esmeralda and Perla barley starch

L.J. Gutiérrez-Osnaya, J.P. Hernández-Uribe, J. Castro-Rosas,

A.D. Román-Gutiérrez, B.H. Camacho-Díaz, H.M. Palma-
Rodríguez, M.L. Rodríguez-Marín, J. Hernández-Ávila, F.A.
Guzmán-Ortiz

PII: S0141-8130(19)40152-9
DOI: https://doi.org/10.1016/j.ijbiomac.2020.01.245
Reference: BIOMAC 14549

To appear in: International Journal of Biological Macromolecules

Received date: 11 December 2019

Revised date: 21 January 2020
Accepted date: 24 January 2020

Please cite this article as: L.J. Gutiérrez-Osnaya, J.P. Hernández-Uribe, J. Castro-Rosas,
et al., Influence of germination time on the morphological, morphometric, structural,
and physicochemical characteristics of Esmeralda and Perla barley starch, International
Journal of Biological Macromolecules(2020), https://doi.org/10.1016/
j.ijbiomac.2020.01.245

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© 2020 Published by Elsevier.

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Influence of germination time on the morphological, morphometric, structural, and

physicochemical characteristics of Esmeralda and Perla barley starch

L. J. Gutiérrez-Osnayaa,b, J. P. Hernández-Uribea, J. Castro-Rosasb, A. D. Román-

Gutiérrez , B. H. Camacho-Díaz , H. M. Palma-Rodríguez , M. L. Rodríguez-Marínd, J.
b c a

Hernández-Ávilae, F.A. Guzmán-Ortizd*.

*Corresponding author: fabiguzman01@yahoo.com.mx, phone: +527717172000 ext. 2514.

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a
Centro de Investigación en Ciencia y Tecnología de Alimentos, Instituto de Ciencias

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Agropecuarias, Universidad Autónoma del Estado de Hidalgo.
b

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Área Académica de Química, Universidad Autónoma del Estado de Hidalgo, Carretera

Pachuca-Tulancingo km 4.5, Ciudad del conocimiento, Mineral de la Reforma, Hidalgo,

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México.
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c
Centro de Desarrollo de Productos Bióticos, Instituto Politécnico Nacional, P.O. Box 24,
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Yautepec 62730, Morelos, Mexico.

d
CONACYT-Universidad Autónoma del Estado de Hidalgo, Carretera Pachuca-
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Tulancingo km 4.5, Ciudad del conocimiento, Mineral de la Reforma, Hidalgo, México.

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e
Área Académica de Ciencias de la Tierra y Materiales, Universidad Autónoma del Estado

de Hidalgo, Carretera Pachuca–Tulancingo, Km 4.5 s/n, Mineral de la Reforma, Hidalgo

C.P. 42184, Mexico.

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Abstract

The aim of this study was to analyzing the impact of germination time on the morphology,

crystallinity, gelatinization and viscosity properties on the starch of Esmeralda and Perla

barley variety. The two barley were germinated for 1 to 8 days, at 26 ºC and 65% relative

humidity. Micrographs showed the presence of pinholes and eroded surfaces. Starch in

Esmeralda was hydrolyzed completely at 8 days of germination. Birefringence was reduced

from day 4, losing molecular structuring of the crystalline area. Morphometric data: fractal

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dimension, area, perimeter, circularity, and roundness decreased significantly along

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germination time in both varieties. The entropy increased significantly, from 0.79 to 10.09

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in Esmeralda and from 0.46 to 7.57 in Perla. Relative crystallinity decreased significantly in
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the Perla from 24.7% to 23.6%. Viscosity peaks were also significantly reduced, pasting

temperature was constant in Esmeralda but in Perla was significantly reduced from 95.43 to
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95.19°C with germination, the gelatinization temperature increased significantly in the

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Esmeralda while in Perla it remained constant. Enthalpy decreased significantly to 75.8%

and 37% in Esmeralda and Perla respectively. The study of germination impact on
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structural and physicochemical properties is important to identify the use of hydrolyzed

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starches in the food industry or others.

Keywords: Barley; starch; germination; morphometric; physiochemical.

1. Introduction

Germination in cereals and seeds is considered as an enzymatic modification tool, because

it generates changes through the use of grain enzymes.

When compared against native ones, germinated grains contain more proteins, minerals,

and vitamins [1]. Recently, germinated grains have garnered attention due to their better

nutritional quality through enzymatic actions. Changes present as a consequence of

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germination are not only functional but also structural, morphological, and

physicochemical.

Elkhalifa et al. [2] determined the influence of sorghum grain germination on functional

properties. They reported a flour with greater emulsifying activity, lower gelation

concentration, and increased functional properties as a consequence of enzymatic activity

during germination. However, these alterations in the functional properties derive from a

structural and physicochemical modification.

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Some studies report that barley grains show thinning of the aleurone layer and cell wall

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degradation during germination. This modifies the endosperm by starch hydrolysis and

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increased enzymatic activity [3]. Sun et al. [4] studied the germination of mutant rice grain
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versus that of native grain. At day 10, there were structural changes in the starch granule of

mutant rice and a total loss of structure in the starch granule of native rice. Germination of
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the mutant rice grain reduces the content of resistant starch, making it easily digestible. You
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et al. [5] used scanning electron microscopy (SEM) to visualize changes in the structure of

starch granule, an increase in porosity, which leads to a decrease in granule crystallinity

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associated to an alteration in the molecular structure of the granule.

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Due to possible modifications in molecules as starch after germination, the functionality of

flours can be different. Therefore, knowing their morphological and structural

characteristics is important for their use in foods. For instance, higher pasting properties are

ideal to thicken foods or gel; otherwise, a lower pasting property is preferable [6]. In

addition, changes in pasting properties can affect the final product of bread, particularly

volume and crumb density depending on starch gelatinization, gelation, and protein

aggregation [7]. It has also been reported that protein digestibility is improved during

germination [8].
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Most of the research on barley has focused on malt treatment. Morphological and structural

studies at different germination in these varieties have not been reported. Studies in

morphometric data have not been reported; however, these data indicate the main

alterations in the morphology of starch. Then, the aim of this study was to determine the

effect of the germination process on the morphology, relative crystallinity, gelatinization

and viscosity properties of isolated starches from Esmeralda and Perla barley. It is

important to understand the impact of germination on the physical and chemical properties

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of crops before they are incorporated to foods as ingredients.

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2. Materials and methods

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Esmeralda and Perla varieties, grown in Apan, Hidalgo, were used. Grains were donated by
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producers. The Esmeralda variety is a 6-row barley, while Perla is 2-row.

2.1 Germination
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The germination process was carried out according to the methodology by Guzmán-Ortiz et
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al. [9], with some modifications. Grains were selected, eliminating damaged, bare and broken

grains and disinfected using 0.05% sodium hypochlorite. Barley grains were soaked in tap
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water (1:2 w/v) at room temperature for 24 h, placed in plastic trays, and moistened to
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saturation. The trays were then placed in a dark germination chamber at 26 ºC and 65%

relative humidity. Grains were disinfected with sodium hypochlorite (0.05%) after 24 h.

Seeds were moistened with water spray every 12 h for 8 days. Only for the microscopy

analysis, samples were taken daily and after 0, 2, 4, 6 and 8 days of germination were

analyzed. Germinated grains were dried until reaching 8% humidity at 40 ºC in an oven for

24 h, approximately. Finally, they were ground in a 2HP Engine 16 in mill (Raymond,

USA) and stored under refrigeration at 4 ºC.

2.2 Starch isolation

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The method by Adkins et al. [10] was used to isolate starch. Germinated grains were

soaked in a regulating solution of sodium acetate (0.02 M) and potassium chloride (0.01 M)

(1:1, v/v) for 16–24 h; the ratio of grain to solution was 1:2 (w/v). The mix was kept under

refrigeration. Germinated grains were drained, washed, and ground. The resulting paste was

sieved through 150, 212, 250, and 500-micron meshes. The extract was centrifuged at 3500

rpm for 15 min and the supernatant was discarded. The precipitate was suspended in a

solution containing 0.1 M NaCl and toluene (7:1, v/v) with constant stirring at room

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temperature for 15 h. The precipitate was centrifuged again at 3500 rpm for 15 min and

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supernatant was discarded. Starch was dried in an oven at 35 ± 3 ºC for 12 h, ground, and

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sieved through a 100-micron mesh to obtain a homogeneous powder.
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2.3 Scanning electron microscopy

Samples were placed in an aluminum holder using double-sided carbon tape and coated
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with gold by sputtering for 3 min. Samples were observed under a scanning electron
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microscope (JSM-6300, Jeol, Japan) at magnifications of 500, 1000, and 2000 at 20 kV.

2.4 Polarized light microscopy

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The starch granules obtained from the germinated and ungerminated grains were examined
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to determine the presence of birefringence through a polarized light microscope (Leitz,

Wetzlar, Germany) using 10x and 40x lenses and equipped with a digital camera (MTI DC-

330, Dage, Chiba, Japan). The samples were placed on slides with coverslips [11]. Images

of hydrated and unhydrated starch were captured with Pixela Image Mixer v 3.0.

2.5 Digital image analysis

The morphology of the starch granule was analyzed, using ImageJ software (V2.31; NIH,

Bethesda, MD, USA), Java, of the two germinated and non-germinated barley varieties;

From the micrographs obtained by scanning electron microscopy (SEM), the following
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morphometric parameters of size, shape and irregularity were obtained: fractal dimension,

entropy, area, perimeter, circularity and roundness [12]. The images were binarized (black

and white) and manually segmented using the "Threshold" tool adjusting the gray levels (0

to 60). The threshold values were generated according to the results of the histogram

analysis and maintained for the analysis of all the groups analyzed.

2.6 Fourier transform infrared spectroscopy (FT-IR)

Once the starch of the two barley varieties was isolated, germination days (0, 2, 4, 6, and 8

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days) were analyzed. The IR-spectrum of the sample was obtained using FTIR

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spectrophotometer (Spectrum GX, Perkin Elmer) following the KBr method (starch/KBr

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ratio 4:1) at 27 ºC, per the methodology by Lian et al. [13]. The mix was pressed to obtain a

pastille and it was analyzed at a resolution interval of 400–4000 cm–1.

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2.7 Differential scanning calorimetry (DSC)
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The thermal properties of the samples were measured using a differential scanning
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calorimeter (DSC Q2000, TA Instruments Inc., New Castle, DE, USA). Samples were

weighed in 40 µL (2 mg) aluminum holders and added distilled water with a syringe (7
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µL). Holders were hermetically sealed and left to rest for 30 min. Samples were heated at
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an interval of 30 at 100 ºC and heating speed of 10 ºC/min. A hermetically sealed holder

was used as initial reference. Initial temperature (To), peak (Tp) or gelatinization

temperature (Tg), final temperature (Tf), and transition enthalpy (∆H) were obtained

directly with a scanning differential calorimeter software [5].

2.8 Rapid visco analyser (RVA)

The pasting properties were determined using RVA4500 (Perten Instrument, Sweden) using

starch of the two varieties of barley. A suspension of 3.5 g (14% moisture basis) starch of

in 25 g of accurately weighed distilled water underwent a controlled heating and cooling

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cycle under constant shear where it was held at 50 °C for 1 min, heated from 50 to 95 °C at

12 °C/min and held at 95 °C for 2 min, cooled to 50 °C at 12 °C/min and held at 50 °C for

2 min. The sample was firstly dispersed for 10 s at a speed of 960 rpm and then a speed of

160 rpm was used throughout. Pasting parameters such as peak viscosity, holding strength

and pasting temperature, were recorded [5].

2.9 X-ray diffraction and relative crystallinity

X-ray diffraction patterns of the starch were obtained with an X-ray diffractometer

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(Equinox 2000, Inel) at 30 KV and 20 mA. The diffractograms were obtained from an

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exploration interval of 4–110 º (2 Ɵ) and a 2.0º/min scanning speed. Relative crystallinity

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was calculated according to the method by Colussi et al. [14] with Origin 9.0 software.
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2.10 Statistical analysis

Results were analyzed with SPSS 16.0 statistical software (IBM, USA). Data were
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expressed as mean and standard deviation in triplicate. ANOVA and Tukey test were used
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to determine the significance between means at different germination times.

3. Results and discussion

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3.1 Scanning electron microscopy

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The morphological characteristics of the starch granules in the germinated and

ungerminated grains of Esmeralda and Perla barley are shown in Figures 1 and 2,

respectively. It was found that the starch granules in the ungerminated barley samples

(Figures 1A and 2A) were adhered to a protein matrix and their surface was smooth oval

and polyhedron shaped. Zhu et al. [15] observed similar morphologies in rice. The starch

granules were released from the protein matrix since day 1 of germination (Figures 1B and

2B). According to Higgins et al. [16], this is likely due to the enzymatic activation taking

place while the barley grains are soaked. The Esmeralda variety showed small pores on the
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surface of starch granules from day 3 to 6 of germination and deformation of granule

morphology (Figure 1D-I). However, pores were observed in the Perla variety until day 5

(Figure 2F). On the other hand, no presence of starch was observed in the Esmeralda

variety at days 7 and 8 of germination (Figure 1H, I). Additionally, bodies of fragmented

proteins and those of different sizes are shown. The morphology observed is constituted by

irregular structures, indicating that the starch was completely hydrolyzed at that time. At

day 8, we observed starch granules of Perla barley that were not completely hydrolyzed and

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whose morphology changed into a rough surface (Figure 2I) due to the

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decompartmentalization of the grain’s tissue. Claver et al. [17] attribute this behavior to the

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increase in enzymatic activity as germination progresses.
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The changes in the starch granules during this process can be related to the enzymatic

activity [17]. It has been reported that amylases are activated when released from the
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endosperm by gibberellins during the steeping of seeds. Some studies indicate that the
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maximum activity of -amylase takes place between days 3 and 5, while that of protease

can start from day 5 of germination [2, 18]. The different changes in the morphology of the
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grain through the germination time are influenced by the variation of the enzymatic activity
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depending on the germination conditions and viability of the seed. Some researchers have

suggested that starch granules of cereals are eroded during germination, showing rough

surfaces and pinholes [5, 19, 20]. Partial hydrolysis of starch is attributed to the erosion of

the granule surface or the digestion of the channels from the selected points on the surface

towards the center. The uneven modification of the surface might be result of variation in

the susceptibility to hydrolytic enzyme activity during germination. The enzyme spreads to

the solid surface and is adsorbed through the sites; finally, the catalytic reaction is produced
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[17]. Adsorption can be affected by granule size, surface characteristics, and minor

constituents, as proteins and lipids on the surface [21].

After the adsorption during the catalytic reaction, starch granules are hydrolyzed by exo or

endocorrosion. Exocorrosion mainly involves the erosion of complete or partial starch

granules by hydrolytic enzymes, while in endocorrosion -amylase activity hydrolyzes the

linkages in the internal region of the substrate, leading to the creation of pores or cavities

[17]. These increase in the transversal section, where they help to spread more enzymes,

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which are finally led to the core of the granule [18].

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Kalita et al. [18] reported a similar behavior of malted rice during germination at 30 and 35

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ºC for 120 h. Micrographs of native rice showed that starch granules had an irregular
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surface without alterations. However, granules of malted rice lost their homogeneity and

showed a rough and eroded morphology. This behavior was attributed to starch hydrolysis
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by hydrolytic enzymes produced during germination. Xu et al. [22] also studied the impact
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of germination on adlay seeds (Coix lacryma-jobi) whose native starch flour was

characterized by a compact and homogeneous structure. The structure was destroyed after
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germination and the process increased as germination progressed. Li et al. [1] reported the
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effect of germination on the structure of brown rice, millet, oat, and sorghum. After 48 h of

germination, the starch granules of these cereals showed an irregular shape, including some

dents and cavities in the surface.

Changes in the surface and the increase in pores are related to the enzymatic action and the

proportion of amylose and amylopectin. Starches with higher proportions of amylopectins

are more susceptible to enzymatic hydrolysis [23]. The proportion of amylopectin could be

higher in Esmeralda barley than in Perla variety since starch degradation was greater in the
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latter. Wu et al. [19] reported that protein bodies disappear due to the action of proteolytic

enzymes activated during germination in starch granules of different rice crops. Then, there

is a maximum proteolytic activity at day 4 of germination, which leaves starch more

exposed to enzymatic hydrolysis.

3.2 Polarized light microscopy

Under polarized light, starch granules show two dark bands, which form a cross in different

directions on the surface. This property is the high order of the structure belonging to

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amylose and amylopectin layers within granules. Morphological changes are greater at days

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2, 4, 6, and 8 of germination. The malt cross was evaluated in these samples and control

(ungerminated sample).
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Figure 3 shows the polarized light micrographs of Esmeralda barley. Starch of

ungerminated barley exhibits a malt cross, indicating there is no damage or modification in

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the starch (Figure 3A). At day 2 of germination, some starch granules show no malt cross
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since they underwent morphological changes as a consequence of enzymatic action. At day

4, more starch granules show no birefringence, indicating the loss of molecular order in the
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crystalline area [24]. The loss of birefringence is more evident as germination progresses.
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At day 6, most of the granules have been modified by enzymatic hydrolysis [17]. As in

Esmeralda barley, ungerminated grains of Perla variety showed malt crosses (Figure 4).

Similarly, as germination time continued, birefringence was lost. At day 8 of germination

(Figure 4E), starch granules were hydrolyzed since the malt cross was not present in all

granules. Figures 5 and 6 show unhydrated starch granules under a polarized light

microscope in bright field. Unlike in scanning micrographs, the presence of pores and

granule deformation is more detailed under polarized light. Starch granules show small

pores from day 2 of germination and, as time elapses, there are more and larger pores. The
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loss of birefringence is related to the concavity observed in SEM. Starch granules lose

resistance to enzymes and increase hydrolysis speed, which leads to loss of crystallinity and

alterations in the granular structure [25, 26]. Rivas et al. [11] reported the morphological

and molecular characterization of native and modified banana starch by oxidation. They

obtained loss in the order of molecule structure in the modified starches.

3.3 Image analysis

Table 1 shows the values of fractal dimension, entropy, area, perimeter, circularity, and

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shape of starch granules of germinated and ungerminated Esmeralda and Perla barley. The

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fractal dimension for both varieties decreases as germination progresses, indicating that

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starch granules are irregular and rough, a behavior associated to the loss in crystallinity
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degree [12]. Esmeralda barley showed no significant difference (p>0.05) between

ungerminated grains and sample germinated for 2 days. However, at day 6 of germination,
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the lowest value was observed. A similar behavior was found in the Perla variety. Espinosa
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et al. [27] reported a similar behavior in rice grains, whose fractal dimension is reduced as

the grain matures. Along with fractal dimension, the irregularity of starch granules
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associated to germination was identified. The fractal dimension of a straight line is

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approximately 1; if the line is sinuous or rough (irregular), the value is higher than 1 [28].

This behavior was observed in both varieties on all days of germination, indicating the

irregularity of the grains. The value for the proportion of a plane occupied by the object of

interest will be approximately 2. The opposite is true when the object is a plane (irregular)

since its values will be lower given that it is an incomplete plane [29]. Entropy behaved

differently from fractal dimension with germination and increased along time. Therefore,

there is a greater degree of molecular disorder in the starch granule as germination time

increases. The Esmeralda variety showed significant changes every day (p<0.05), while
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Perla variety only exhibited changes from day 4 of germination. Esmeralda barley showed a

greater entropy, confirming the results obtained from micrographs (Figure 1) where higher

hydrolysis was observed in starch granules.

Changes in fractal dimension and entropy are also correlated to the area and perimeter in

morphology. The germination process had a significant effect on the area of starch

granules. In both varieties, starch granules showed a smaller area according to germination

time. Areas in Esmeralda barley were larger than those in the Perla variety, which could be

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due to the chemical composition of barley grains during their development. They might be

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exposed to different drastic changes in temperature or relative humidity [30, 31]. The area

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in Esmeralda barley decreased 4.5 times by day 6 of germination, while that in Perla barley
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was reduced 3.2 times by day 8 (Table 1). This decrease is associated to hydrolysis from

the external to the internal granule and disorder in linkages as a consequence of

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germination. The perimeter also decreased between 1.6 and 1.4 times in Esmeralda and
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Perla varieties, respectively. This behavior was due to hydrolysis in the starch caused by

amylases present during the biochemical process of germination. There was rupture and
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damage to the morphology of starch granules, as observed in the micrographs obtained

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from both varieties of barley (Figures 1 and 2). Circularity and roundness also indicate the

shape of starch granules. If the circularity value is approximate 1, the object is spherical

and, if it is higher than one, the object is an ellipse. The object is circular when the

roundness value is equal to 1 and it loses circularity when it is lower than 1 [27]. Circularity

results for Esmeralda barley were 0.81 mm in ungerminated grain, reaching 0.42 mm at day

6 of germination; differences were significant (p<0.05). The Perla variety started with

circularity values of 0.90 mm and reached 0.43 mm at day 8 of germination, showing

significant differences (p<0.05). Perla barley exhibited more spherical starch granules at the
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beginning. This characteristic proved there is a loss of the spherical or oval shape that

barley starch granules must have as germination time elapses due to enzymatic hydrolysis.

Roundness parameters in both varieties of barley showed similar values in ungerminated

grains: 0.85 mm and 0.86 mm for Esmeralda and Perla, respectively. In addition, this

characteristic is lost during germination. Perla barley showed higher values, suggesting that

its structure is more complex and thus delays the enzymatic attack, leading to a lesser

damage to its morphology. At day 8 of germination, the starch was not completely

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hydrolyzed, unlike that of Esmeralda barley.

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This could confirm alterations in the shape and size of Perla barley when it undergoes a
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germination process. However, the physicochemical properties of its starch granules

improve at the same time.

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3.4 Infrared spectrum

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Figures 7A and 7B show the infrared spectra of the starches obtained from germinated and

ungerminated barley of both varieties. A hydrogen stretching band was observed at 2980
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cm–1, corresponding to the carbon bond characteristic in the presence of carbohydrates [32,
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33]. This vibration was not affected by germination in any of the varieties. The band at

1652.92 cm–1 corresponds to the CO stretching of C-1 [34, 35]. The peaks adjacent to 1450

cm–1 and 1380 cm–1 are the result of C-H angle vibration, which did not show any

alterations. Absorption at 1074 cm–1 and 1019 cm–1 indicates COC and CO bonds of a D-

glucose ring; then, this region indicates the presence of glycosidic bonds and

monosaccharide structures [36]. The Esmeralda variety showed a slight increase in these

bands from day 6 of germination. This was associated to the degree of starch hydrolysis in
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reducing sugars by - and β-amylases during the germination process [18]. However, the

Perla variety did not exhibit the same behavior.

On the other hand, was observed signals in the interval 900–1280 cm–1 corresponding to the

C-O stretching bond. Some authors report that the bands between 1047–995 cm–1 are

associated to molecular order and the crystalline region of starch [37, 38]. Loss of

crystallinity may also be associated with loss of signal to 995 cm–1, by disassociation and

unfolding of some hydrogen bonds within the crystalline region helices of the starch [39].

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The results obtained by infrared spectroscopy in this region were similar to those reported

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by Tarr et al. [40] in barley malt. In addition, You et al. [5] studied the impact of

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germination on rice starch and found reduced crystallinity as germination progressed, based

on the observation of bands at 1047 cm–1 and 1022 cm–1 corresponding to the crystalline
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and amorphous regions, respectively.
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3.5 Crystallinity
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Relative crystallinity values calculated from diagrams of X-ray diffraction are shown in

Table 2. Esmeralda barley showed no significant effect (p>0.05) along germination;

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however, it exhibited lower crystallinity values when compared versus the Perla variety.
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This could be related to the greater starch hydrolysis observed in SEM micrographs. In

Perla barley, crystallinity slightly decreased from day 4 of germination, possibly because of

starch amylose and amylopectin degradation by enzymatic activity during germination [1,

18]. Crystallinity is directly associated to the proportion of amylopectin [41]. Long chains

of starches organize crystalline regions and, when degraded in germination, the content of

relative crystallinity is reduced [1]. You et al. [5] reported that a long chain of amylopectin
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generates greater relative crystallinity. Accordingly, the reduced relative crystallinity can be

attributed to the presence of shorter branched chains and their greater hydrolysis.

3.6 Pasting properties

The pasting properties of starch obtained from germinated and ungerminated grains of

Esmeralda and Perla barley are presented in Table 3. The maximum and holding strength

decreased significantly (p<0.05) along germination. The viscosity of ungerminated grains

of both varieties was higher than that of germinated grains at day 2 since -amylase is

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inactive during this stage. At day 8 of germination, this property was not detected in

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Esmeralda variety given that the starch was totally hydrolyzed, as shown in Figure 1.

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The tendency of viscosity to decrease along germination is likely the consequence of starch

granules losing resistance to swelling because of higher -amylase activity that reduces
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viscosity [42]. Additionally, the total content of starch is reduced by the enzymatic
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hydrolysis of amylase and proteins by proteases during germination [43, 19].

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Starch from Esmeralda barley showed the greater morphological changes in the grain

surface (Figure 1). These data correlate with viscosity values since this variety showed
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lower levels when compared against Perla barley. Li et al. [1] reported a similar behavior in
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sorghum and millet starches. Uthumporn et al. [44] report that a decrease in viscosity is due

to the porous structure, which makes starch granules more susceptible to hydrolyzation.

Zhu et al. [45] reported that amylase activity in the grains contributes to the reduction of

maximum viscosity. Quadir et al. [46] and Xu et al. [47] published preliminary results of

Kashmiri and brown rice, both native and germinated. The pasting temperature of

Esmeralda barley showed no significant effects (p>0.05) and was constant during
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germination. Contrastingly, the temperature in the Perla variety was reduced at day 4 of

germination.

Similar tendencies have been reported by Xu et al. [47] and Wu et al. [19] in germinated

rice. Chinma et al. [48] found a slight reduction in pasting temperature of germinated

brown and yellow tiger nut. The difference in behavior is influenced by the initial

composition of barley grains (starch, lipid, and protein content, among others), the

amylose/pectin proportions, and the enzymatic activity.

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3.7 Thermal properties

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The thermal properties of starch from both varieties were analyzed and the results are

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shown in Table 4. The gelatinization temperature in the Esmeralda variety was significantly
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increased (p<0.05) from day 4 of germination, while that of the Perla variety decreased at

day 2 and rose at day 4 of germination. The decrease could be due to lipid degradation in
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the early stages of germination [19]. Lipids cover the surface of the starch which limits the
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swelling of the starch granules [49]. Furthermore, the relation amylose/amylopectin might

also affect the behavior of gelatinization [50].

ur

On the other hand, the constant increase in gelatinization temperature of the Esmeralda
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variety is likely caused by the sugars accumulated during germination [19]. It has also been

reported that the presence of some amino acids and protein hydrolysates can increase the

temperature of starch gelatinization [22]. Li et al. [1] reported a similar behavior; there was

an increase in gelatinization temperature of rice, sorghum, and millet at 24 and 48 h of

germination. Wu et al. [19] reported similar temperatures and increases in germinated

flours of several varieties of rice. Xu et al. [22] evaluated adlay seed (Coix lacryma-jobi)

germinated for 12, 24, 36, 48, and 60 h and also found increased temperature.

Contrastingly, the gelatinization enthalpy was reduced in both varieties. In Esmeralda

 Journal Pre-proof

barley, it was reduced from 6.46 to 1.56 J/g at day 6 of germination, while it decreased

from 7.93 to 4.96 J/g at day 8 of germination in the Perla variety. The molecular structure

of starch in amylopectin chains affects gelatinization enthalpy [51], which represents the

loss of double helix order in the crystalline and amorphous regions [52].

During germination, some hydrogen bonds uniting adjacent double helices are ruptured,

which contributes to a lower proportion of longer branched chains, leading to a decrease in

gelatinization enthalpy [53, 1]. The type of sugars could also affect the behavior of

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enthalpy. Baek et al. [54] reported that certain sugars reduced gelatinization enthalpy,

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mainly monosaccharides. In addition, Esmeralda barley showed lower enthalpy values

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since, morphologically, its starch exhibited more hydrolysis (Figure 1). Then, we deduced
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this variety has a higher concentration of monosaccharides than Perla barley.

4. Conclusion
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The data in this study demonstrated that time in the germination process greatly affects the
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morphology, structure, and physicochemical structure of barley starch. The morphometric

parameters were lower along the germination process, revealing the change in starch
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granule morphology in time. There is a greater degree of molecular disorder in the starch
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granule as the germination time increases since the entropy values were higher in both

varieties and the malt crosses were lost. The Esmeralda variety showed greater

susceptibility to microstructural changes than the Perla variety, suggesting that Esmeralda

has greater enzymatic activity. Viscosity was reduced along germination, while pasting

temperature was constant and only slightly reduced in Perla barley, likely associated to the

amylose/amylopectin relationship. This study showed the impact of germination time on

starch hydrolysis on structural and physicochemical properties, which are important for

identifying the use of hydrolyzed starches by a simple process in the food or other industry.
 Journal Pre-proof

Acknowledgements

This research manuscript was supported by Catedras CONACyT project 1232. All authors

possess no conflicts of interest.

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Table 1. Morphometric parameters of germinated and ungerminated barley starch

granules

Esmeralda variety barley

Fractal Perimeter Circularit

Days Entropy Area (mm2) Roundness
dimension (mm) y

S/G 2.44±.011a 0.79±.012d 6738.89±553.37a 319.91±7.07a 0.81±0.05a 0.85±0.07a

2 2.23±0.17ab 2.82±0.77c 4589.10±276.45b 310.93±5.08ab 0.74±0.05a 0.78±0.04a
4 1.95±0.08b 6.52±0.62b 3082.68±307.47c 287.63±11.80b 0.72±0.05a 0.63±0.04b
6 1.20±0.12c 10.09±0.18a 1493.15±260.73d 197.60±11.33c 0.42±0.09b 0.29±0.06c

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8 Not detectable

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Perla variety barley

Fractal Perimeter Circularit

Area (mm2)
Days
dimension

2.35±0.07a
Entropy

0.46±0.11d
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5952.43±267.22a
(mm)

299.94±11.65a
y
Roundness

0.90±0.04a 0.86±0.07a
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S/G
2 2.08±0.16a 1.05±0.09cd 5246.37±228.02b 275.49±6.78ab 0.82±0.05a 0.80±0.06ab
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4 1.74±0.14b 1.37±0.16c 4404.72±319.73c 247.30±14.20bc 0.63±0.04b 0.68±0.03b

6 1.57±0.11b 5.13±0.35b 2755.24±233.90d 224.79±16.48cd 0.56±0.07bc 0.50±0.06c
1.16±0.11c 7.57±0.62a 1843.36±157.40e 200.04±8.54d 0.43±0.05c 0.37±0.03c
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8
The results are the average of three determinations ± the standard deviation.
a-b letters indicate comparison of means between the same sample.
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Samples with the same letter did not present significant difference using Tukey's test (P <0.05).
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Table 2. Relative crystallinity of germinated and ungerminated starch granules

Esmeralda Perla
Days
(%) (%)
S/G 22.67±0.57ª 24.70±0.64a
2 22.24±0.57ª 23.86±0.18ab
4 22.10±0.37ª 23.29±0.34b
6 21.87±0.54ª 22.71±0.49b
8 Not detectable 23.60±0.67b
The results are the average of three determinations ± the standard deviation.
a-b letters indicate comparison of means between the same sample.

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Samples with the same letter did not present significant difference using Tukey's test (P <0.05).

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Table 3. Paste properties of germinated and ungerminated barley starch granules.

Variety Peak Viscosity Pasting temperature
Holding strength (cP)
Esmeralda (cP) (°C)
S/G 2730.50±201.50a 2630.50±101.50a 95.33±0.20a
2 2338.50±23.50b 2115.50±59.50b 95.38±0.03a
4 1880.50±70.50c 1331.50±50.50c 95.20±0.10a
6 510.50±20.50d 415.50±47.50d 95.28±0.23a
8 Not detectable
Peak Viscosity Pasting temperature
Perla Holding strength (cP)
(cP) (°C)
S/G 3101.00±25.00a 2813.00±60.00a 95.43±0.02a

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2 2443.00±48.00b 1986.50±34.50b 95.43±0.02a
4 1087.00±106.00c 780.00±56.00c 95.33±0.02b

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6 715.00±70.00d 415.00±20.00e 95.29±0.03b
8 828.50±32.50d 589.50±42.50d 95.19±0.02c

-p
The results are the average of three determinations ± the standard deviation.
a-b letters indicate comparison of means between the same sample.
Samples with the same letter did not present significant difference using Tukey's test (P <0.05).
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Table 4. Thermal properties of germinated and ungerminated barley starch granules.

Variety Gelatinization temperature Enthalpy

Esmeralda (°C) ΔH (J/g)
S/G 61.36±0.24b 6.46±0.03a
2 61.37±0.14b 6.39±0.07a
4 63.11±0.47a 2.30±0.42b
6 63.40±0.31a 1.56±0.28c
8 Not detectable
Gelatinization temperature Enthalpy
Perla
(°C) ΔH (J/g)

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S/G 61.83±0.01ab 7.93±0.54a
61.73±0.12b 6.58±0.19b

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2
4 62.55±0.50a 6.52±0.02b
6
8
62.22±0.25ab
62.08±0.20ab -p 6.50±0.04b
4.96±0.34c
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The results are the average of three determinations ± the standard deviation.
a-b letters indicate comparison of means between the same sample.
Samples with the same letter did not present significant difference using Tukey's test (P <0.05).
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Figure captions
Fig. 1. Micrographs of Esmeralda barley grains: SD) Ungerminated barley, D1) 1 day of
germination, D2) 2 days of germination, D3) 3 days of germination, D4) 4 days of
germination, D5) 5 days of germination, D6) 6 days of germination, D7) 7 days of
germination, D8) 8 days of germination.

Fig. 2. Micrographs of Perla barley grains: SD) Ungerminated barley, D1) 1 day of
germination, D2) 2 days of germination, D3) 3 days of germination, D4) 4 days of
germination, D5) 5 days of germination, D6) 6 days of germination, D7) 7 days of
germination, D8) 8 days of germination.

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Fig. 3. Barley starch granules of the Esmeralda variety through polarized light: SG)

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Ungerminated barley, D2) 2 days of germination, D4) 4 days of germination, D6) 6 days of
germination. (40X, hydrated sample).
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Fig. 4. Barley starch granules of the Perla variety through polarized light: SG)
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Ungerminated barley, D2) 2 days of germination, D4) 4 days of germination, D6) 6 days of
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germination, D8) 8 days of germination. (40X, hydrated sample).

Fig. 5. Barley starch granules Esmeralda: SG) Ungerminated barley, D2) 2 days of
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germination, D4) 4 days of germination, D6) 6 days of germination. (40X, clear field,
sample without hydrating).
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Fig. 6. Barley starch granules Perla: A) Ungerminated barley, D2) 2 days of germination,
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D4) 4 days of germination, D6) 6 days of germination, D8) 8 days of germination. (40X,
clear field, sample without hydrating).

Fig. 7. Infrared Spectrum of Esmeralda (A) and Perla (B) barley starch: SD) Ungerminated
barley, D1) 1 day of germination, D2) 2 days of germination, D3) 3 days of germination,
D4) 4 days of germination, D5) 5 days of germination, D6) 6 days of germination, D7) 7
days of germination, D8) 8 days of germination.
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Author contributions

L. J. Gutiérrez-Osnaya: Investigation, Validation, Formal analysis

J. P. Hernández-Uribe: Conceptualization, Methodology, Validation, Writing - Review

& Editing

J. Castro-Rosas: Resources, Visualization

B. H. Camacho-Díaz: Methodology, Software, Writing - Review & Editing

A. D. Román-Gutiérrez: Resources, Visualization

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H. M. Palma-Rodríguez: Methodology, Resources

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J. Hernández-Ávila: Software, Data Curation

M. L. Rodríguez-Marín: Formal analysis, Writing - Review & Editing

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F.A. Guzmán-Ortiz: Conceptualization, Methodology, Investigation, Writing - Original
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Draft, Supervision, Project administration
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Highlights
 Germination modifies the morphometric parameters of the starch granule in barley
 Esmeralda showed greater susceptibility to microstructural changes than the Perla
 Entropy increases and the malt cross is lost in germinated barley starch.
 The crystallinity and gelatinization temperature change with the germination time.

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