


































Food Science and Nutrition Studies 

ISSN 2573-1661 (Print) ISSN 2573-167X (Online) 

Vol. 3, No. 4, 2019 

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122 
 

Original Paper 

Hydrothermal Treatment to Remove Tannins in Wholegrains 

Sorghum, Milled Grains and Flour 

María del Rosario Acquisgrana1, Laura Cecilia Gómez Pamies1 & Elisa Inés Benítez1,2* 

1 Departamento de Ingeniería Química, Facultad Regional Resistencia, QuiTEx-Universidad 

Tecnológica Nacional, French 414, 3500 Resistencia, Chaco, Argentina  

2 Facultad de Ciencias Exactas y Naturales y Agrimensura, IQUIBA-NEA, CONICET, Universidad 

Nacional del Nordeste, Av. Libertad 5460, 3400 Corrientes, Corrientes, Argentina 

* Elisa Inés Benítez, Departamento de Ingeniería Química, Facultad Regional Resistencia, 

QuiTEx-Universidad Tecnológica Nacional, French 414, 3500 Resistencia, Chaco, Argentina 

 

Received: October 7, 2019     Accepted: October 18, 2019     Online Published: October 30, 2019 

doi:10.22158/fsns.v3n4p122        URL: http://dx.doi.org/10.22158/fsns.v3n4p122 

 

Abstract 

Pigmented sorghum with high content of tannins were studied in this work. Tannins bind to proteins 

and reduce their availability. A hydrothermal treatment was carried out to reduce tannins. A control 

sample of non-pigmented pericarp variety was used. After the treatment, grains were milled, and a part 

was separated for wholegrain flour elaboration. Several determinations were done after treatment: 

tannins (T), total antioxidant capacity (TAC) and total polyphenols (TPP) content. TPP and TAC in 

wholegrain pigmented sorghum were 3.9 to 12.3 and 2.3 to 3.5 times higher than those of 

non-pigmented sorghum, respectively. In all sorghum varieties the extractions of TPP decreased with 

milling. TAC in flour increased 3.3 times the initial value for non-pigmented sorghum, whereas for the 

other sorghum samples it increased slightly from 1.1 to 1.3 times the initial value. In flours there was a 

noticeable reduction in T, with respect to the wholegrain. It was possible to conclude that the 

hydrothermal treatment allowed lower levels of tannins than those established in the Codex for both 

wholegrain sorghum and flour. This reduction makes it possible to obtain flour which may be suitable 

for food processing and the recovery of tannins for other uses.  

Keywords 

Steeping, Annealing, Milling, Sorghum, Tannin 

 

 

 



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1. Introduction 

Sorghum (Sorghum bicolor (L.) Moench) is the fifth most important cereal crop in the world after 

wheat, rice, corn and barley (Singh et al., 2011), being an excellent source of energy used for both 

animal and human feed (Carvalho Teixeira et al., 2016). The world consumption of this cereal is 

considerable (Althwab et al., 2015) and it is probably due to the ability of the grain to grow over 

extensive agro-ecological zones (Girard et al., 2018; Taylor et al., 2014). This cereal has nutrients 

common to all varieties, including several minerals, vitamins and amino acids (Althwab et al., 2015). 

The presence of polyphenol in the grain is typical in all varieties. The antioxidant level of polyphenols 

in sorghum is higher than in any other cereal analyzed (Rao et al., 2018). The presence of polyphenols 

in the sorghum grain provides natural protection against microorganism and insect attacks 

(Chandrashekar & Satyanarayana, 2006).  

The levels of phenols and antioxidant activity are highest when sorghums have secondary purple/red 

plant color; a black or dark red, thick pericarp and a pigmented testa (Dykes et al., 2005). In pigmented 

sorghum, condensed tannins, belonging to the group of polyphenols, become important. In this variety, 

a positive correlation between total phenolic content and proanthocyanidin, flavan-4-ols and 

3-deoxyanthocyanidins (condensed tannins) has been reported. In the same study, 55% of polyphenols 

correspond to proanthocyanidin 18% to flavan-4-ols and 7.5% to 3-deoxyanthocyanidins (Dicko et al., 

2005). From this previous study, analyzing tannin content in pigmented sorghum through polyphenol 

determination leads to a good estimation. Such hypothesis is evaluated in this new study.  

Condensed tannins have a negative impact on sorghum flours because they reduce the digestibility of 

many nutrients, which can affect animal productivity and health (Awika & Rooney, 2004). Its main 

effect on nutritional value is reducing the digestive availability of protein and starch (Aguiar Moraes et 

al., 2015). However, there is a tendency to use milled wholegrain (Van der Kamp and Lupton, 2013), 

because this type of food may be suitable for diets among people with type 2 diabetes, it is proven that 

polyphenols in sorghum bind to digestive enzymes, specifically alpha-amylase and retard the 

degradation of starch into glucose, attenuating hyperglycaemia (Links et al., 2015). Therefore, the 

hydrothermal treatment proposed in this work would has a double benefit, since on one hand it would 

reduce the tannins in flours, where they are not desired due to their coloration, astringency and 

reduction of protein and starch availability (Links et al., 2015), and on the other hand their separation 

by means of a suitable solvent allowing the use of them as an additive in other foods or as nutraceutical, 

to reduce type 2 diabetes. 

The production of wholegrain flour consists of grinding the whole grain to take advantage of the 

nutrients found in the pericarp and fibers thus improving the gastrointestinal tract health and reducing 

the incidence of chronic diseases (Van der Kamp & Lupton, 2013). The milling of sorghum could cause 

contact of the pericap polyphenols and proteins from the inside of the grain and reduce protein 

availability. On the other hand, the Codex Alimentarius Standard states that wholegrain and flour of 

sorghum cannot contain more than 0.5% and 0.3% tannins, respectively (Codex Alimentarius 



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Commission (CAC), 2018). 

Previous studies proposed water treatment to reduce the content of polyphenols present in the pericarp 

of the wholegrain, to take advantage of polyphenols for other uses in food, and to obtain flour with 

better starch and protein availability (Acquisgrana et al., 2016). After treatment there is still enough 

polyphenols retaining their antioxidant capacity. For that reason, the purpose of this work is to quantify 

the amount of the remaining polyphenols in the grain after treatment, after grain milling and in final 

flour. However, in this work both determination of tannins and polyphenols was done, because tannins 

are the real problem in food.  

The method to quantify the concentration of polyphenols in cereals is not direct, because it is required 

to extract them from the food matrix and in many cases, extraction is incomplete depending on the 

solvent used (Tufan et al., 2013). For a better comparison between different types of solvents, tannins 

in flour is done both, with water and a methanolic extraction. 

It is interesting to quantify the residual antioxidant capacity after extraction, because it is an attractive 

quality in food and it is associated with the presence of polyphenol, then an important loss of this 

property with the hydrothermal treatment applied to wholegrain is expected. The cupric ion reducing 

antioxidant capacity (CUPRAC) method has been applied to cereals and has proved to be a reliable 

determination (Tufan et al., 2013); therefore, this is the method used in this work. 

 

2. Material and Methods 

2.1 Steeping 

Five samples of sorghum with high content of tannin were obtained from the Experimental Agricultural 

Station- National Institute of Agricultural Technology (INTA), Argentina. Four of the samples were red 

or brown sorghum (DK 61T, DOW 108, TOB 60T and Malón– simplified nomenclature: DK, DW, T 

and M). The other sample was non-pigmented sorghum, named Blank sample (B). Total tannin 

concentration for each sample was obtained from extractable tannins during hydrothermal treatment 

and the second extraction used in this study. Total tannin concentrations were summarized in Table 1.  

 

Table 1. Total Tannin Concentration of Each Wholegrain Sorghum Sample 

SAMPLE 
UMS 

mg/kg UMS 

B 1220 ± 21 

DK 7577 ± 54  

DW 9252 ± 60 

M 4443 ± 26 

T 9683 ± 51 

Data are mean values ± standard deviation 



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Sorghum grains (50 g) of each sample were steeped in 100 ml of sodium hypochlorite (NaOCl) 

solution, containing 0.5% (v/v) available chorine. The procedure was done at 25ºC during 18 h 

(Acquisgrana et al., 2017). The samples were washed to eliminate the NaOCl solution and 100 ml of 

water was added. The preparation was incorporated to a heat bath at 75ºC for 60 min (Acquisgrana et 

al., 2016), a stage called “annealing” (Singh et al., 2011). Finally, the samples were dried for 12 h at 

60ºC. 

2.2 Milling  

After annealing and extracting polyphenol, each sample was divided in three samples: unmilled 

sorghum (UMS), milled sorghum (MS) and flour (F). The MS and F samples were milled with a 

two-roller mill (CIBART, Argentina) with a separation of 0.5 mm between rollers. MS samples went 

through the mill once; F samples underwent the same process eight times. F samples were screened 

through a 500 m mesh (ASTM 35) to obtain fine flour, according to CAC.  

2.3 Polyphenol Extraction 

A sample of UMS, MS and F was weighed, and polyphenols were extracted with double the amount of 

water. Each preparation was incorporated to a heat bath at 75ºC for 120 min. Samples were taken every 

30 minutes to analyze total polyphenols (TPP) and total antioxidant capacity (TAC).  

2.4 Measures 

TPP were estimated using the Folin-Ciocalteu method (Singleton et al., 1999) and tannis (T) were 

estimated using the HCl- vainillin midific method (Price et al., 1978). Both methods were expressed in 

mg catechin/Kg of solid matter, UMS or F. TAC was estimated with the CUPRAC method (Özyürek et 

al., 2011) and expressed as mmol trolox equivalents (mmol TE/Kg of solid matter, UMS or F) (Tufan et 

al., 2013). Measurements were carried out in triplicate. 

2.5 Statistical Analysis 

Mean values were calculated, and the software Infostat (2002) was used to analyze variance. Tukey test 

was carry out at the 0.05 significance level. 

 

3. Results and Discussion 

3.1 Polyphenol Determination 

In all UMS samples, it was observed that even after treatment to reduce the content of polyphenols, 

concentration was still significant (Figure 1). Polyphenols were extracted using different solvents: 

water and methanol. Table 2 shows the final concentration of polyphenols and tannins for each sample 

at the end of the extraction process. Total values correspond to extraction at 120 minutes. The same 

table shows the values for the extraction of tannins in aqueous solution and methanolic solution. 

For UMS (Figure 1), it is observed that pigmented sorghum T contains more polyphenols than the rest 

of the samples (0.40%), while non-pigmented sorghum shows the lowest value (<0.03%). For UMS, 

extractable polyphenols corresponds to those found in the pericarp. In all the UMS analyzed a 

second-order polyphenol extraction kinetics could be obtained, which may be modeled with a quadratic 



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polynomial similar to those previously obtained by Acquisgrana et al. (2016). This fact suggests that 

the treatment could continue for longer than the expected 60 minutes, from previous results 

(Acquisgrana et al., 2016). Sorghum with lower tannin content was obtained with longer extraction 

time, but as it will be seen in the MS and F samples, it would not be necessary since the final 

concentrations were adequate according to the Codex. On the other hand, an excessive reduction may 

cause a greater loss of antioxidant capacity, which is not be desirable for food. Furthermore, in previous 

studies it has been observed that the implementation of the proposed treatment greatly improves 

availability of soluble proteins (Acquisgrana et al., 2017). 

 

 

Figure 1. Polyphenol Extraction vs. Extraction Time for Unmilled Sorghum (UMS) 

 

Table 2. TPP, T-WE and T-ME and AC after 120 min of Extraction for UMS, MS and F Samples 

  
UMS 

mg/kg UMS 

MS 

mg/kg UMS 

F 

mg/kg F 

TPP 

B 324 ± 31 377 ± 9 145 ± 47 

DK 3119 ± 11 1515 ± 13 1194 ± 21 

DW 3156 ± 62 1710 ± 50 983 ± 14 

M 1255 ± 41 1008 ± 3 1179 ± 41 

T 3977 ± 36 1537 ± 12 1068 ± 39 

T-WE 

(Water extraction) 

B 440 ± 22 608 ± 23 180 ± 13 

DK 2622 ± 16 3150 ± 38 2787 ± 30 

DW 2828± 9 2900 ± 39 2056 ± 43 

M 1093 ± 25 2218 ± 26 2181 ± 35 

T 3417 ± 55 3471 ± 52 2861 ± 28 



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T-ME (Methanolic 

extraction) 

B  640 ± 38 284 ± 18 

DK  2678 ± 56 2613 ± 32 

DW  3524 ± 83 2144 ± 71 

M  1790 ± 45 2258 ± 55 

T * 3375 ±72 1930 ± 39 

AC** 

mmol TE/kg 

B 1.2 ± 0.2 2.9 ± 0.4 4.0 ± 0.4 

DK 4.2 ± 0.4 5.2 ± 0.2 4.7 ± 0.4 

DW 3.8 ± 0.1 5.5 ± 0.1 4.2 ± 0.1 

M 2.7 ± 0.1 4.4 ± 0.3 3.4 ± 0.3 

T 3.5 ± 0.2 6.1 ± 0.3 5.1 ± 0.2 

* Determination of T-ME in UMS was not done. Data are mean values ± standard deviation 

** AC units are mmol TE/kg instead of mg/kg  

 

Figure 2 shows the extraction of polyphenols for MS. It is observed that practically in all samples, the 

values are stabilized at 90 minutes. The concentrations of polyphenols in MS samples of all pigmented 

sorghums showed lower values than the UMS samples: DK was reduced in 51.4%, DW in 45.8%, M in 

19.6% and T in 61.3% in relation to UMS. White non-pigmented sorghum presented an increase with 

respect to UMS by 16.4% (Table 2).  

The decrease of polyphenols in all sorghum varieties could be directly linked with their interaction with 

proteins. When the grain is milled, polyphenols could interact with proteins and could not continue to 

be extracted, which does not necessarily imply the reduction of the proteins, because they continue in 

the ground matrix.  

In the flour samples (F), since they are completely ground and have been separated from the pericarp 

by sieving, extraction speed is higher than for UMS and MS samples, reaching stability within the 

initial 30 minutes and remaining invariable the rest of the time, therefore only the final value of 

extraction is indicated in Table 2. Since most polyphenols have been extracted during the annealing 

process and the retention of most of the pericarp and germ during sieving, the flours of all the variety 

present a lower concentration of polyphenols than the UMS and MS. Extraction stability after 30 

minutes at 75ºC could indicate the appropriate time to extract all the polyphenols present in the F 

samples. 

Previous studies have reported the high TAC present in sorghum grains, regardless of their variety 

(Dlamini et al., 2007). This previous study considered pigmented sorghums with and without tannins. 

However, it is known that the presence of tannins confers the greatest antioxidant capacity in sorghum 

varieties, due to the presence of proantocyanidin and other condensed tannins (Rao et al., 2018). For 

example, in the case of proanthocyanidin, mean values of 9400 mg/kg for the red variety, against 1300 

mg/kg for the white variety have been reported. Furthemore, the proanthocyanidin levels were 



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positively correlated with the total phenolic content. Other examples and quantities can be comparated 

in Bröhan et al. (2011).  

 

 

Figure 2. Polyphenol Extraction vs. Extraction Time for Milled Sorghum (MS) 

 

Other previous studies have reported a strong correlation between the content of polyphenols and 

tannins (Dicko et al., 2005). This observation could be verified in the present report work (Figure 3), 

but only in whole sorghum the content of tannins is lower than that of polyphenols, finding the linear 

adjustment that is reported in Table 3, with the setting parameters of Equation 1: 

𝑇𝑃𝑃 = 𝑎 ∙ 𝑇                                   (1) 

 

Table 3. Setting Parameters for Eq. (1) Correlation of Tannins and Polyphenols in UMS, MS and 

F, with Water (WE) and Methanolic (ME) Extraction 

T a R2 

UMS-WE 00.91a 0.985 

MS-F-WE 2.11b 0.914 

MS-F-ME 2.02b 0.949 

Mean in same row in different lowercase are significantly different (p<0.05). 

 

From the linear adjustments through the origin for UMS samples, it is obtained that 91% of the 

polyphenols correspond to tannins. However, a strong correlation between tannins and polyphenols is 

possible for MS and F samples, but tannins exceed more than twice the content of polyphenols, 

obtaining no significant differences between both extractions, with water or methanol. There were also 

no significant differences between F and MS samples (Table 3). Probably, no significant differences are 

observed due to the high temperature used for the different types of solvent. The main difference 



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between tannins and polyphenols may be the limitation of the Folin-Ciocalteu method (Singleton et al., 

1999) which does not allow adequate quantification of condensed tannins. Recent studies provide more 

evidence on this behavior where a similar relationship between tannins and popyphenols was found 

(Adetunji et al., 2015). In all cases the tannins values found are much lower than that established in the 

CAC 0.5% for wholegrains and 0.3% for flours, therefore with the treatment proposed by Acquisgrana 

et al. (2016) it is possible to obtain a flour suitable for human consumption from the varieties of 

colored sorghum studied and that can be used for the production of gluten-free foods for people with 

celiac disease.  

 

 

Figure 3. Correlation between Tannins (T) and Total Polyphenols (TPP) in UMS, MS and F 

Samples. Full Lines Represent Eq. (1) with Constant Values in Table 2. Vertical Bars Represent 

the Standard Deviation in Each Value 

 

3.2 Antioxidants 

TAC determination in Table 2 shows that pigmented sorghum contain a greater quantity of antioxidants 

than non-pigmented sorghum in the UMS. In all cases, the TAC is the highest in the UMS samples, and 

the pigmented varieties show higher TAC values than the non-pigmented sorghum variety. This is a 

nutritional advantage of the wholegrain of treated pigmented sorghum since it is possible to reduce the 

content of tannins to suitable levels according to the CAC and that preserve an antioxidant capacity 

greater than the white variety. However, during flour production, the pericarp part containing most of 

the fiber and probably the tannins, is separated with the sieving process, leaving the flour of pigmented 

sorghum with an TAC similar to the non-pigmented variety (Table 2). In the case of flour, it is observed 

that in all cases the values obtained are lower than those found by Tufan et al. (2013) for the aqueous 

extract of 18.28 mmol TE/kg Barley, Rye 8.64, Wheat 4.31 and 7.51 Oat, with the same CUPRAC 

method. It is interesting to show that the previous cereal did not undergo sieving, and probably in this 

samples the TAC will be reduced. However, the values of TAC obtained in the sorghum samples are of 

the same order of magnitude obtained with the other cereals studies and similar to milling wheat.  



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The reduction in the TAC is due to the initial annealing procedure to extract polyphenols. The 

methodology used in this work and in the previous studies (Acquisgrana et al., 2016) can be used to 

regulate the content of tannins to meet the requirements of the CAC and present a residual antioxidant 

capacity that makes it attractive to obtain products with the selected flour. 

 

4. Conclusion 

From the result obtained, it was possible to conclude that the treatments of steeping and annealing 

allowed lower levels of tannins than those established in the Codex for both UMS and F samples. It 

may also be possible to handle this reduction to improve the antioxidant capacity of flour. This 

reduction makes it possible to obtain flour, from pigmented varieties, which may be suitable for food 

processing and the recovery of tannins for other uses.  

 

Acknowledgments 

The authors thank the Facultad Regional Resistencia-Universidad Tecnológica Nacional and the 

Consejo Nacional de Investigaciones científicas y Técnicas (CONICET) for their financial support, and 

the Estación Experimental Agropecuaria del INTA “Las Breñas” for their contribution of experimental 

samples. Special thanks to Mr. Julio Osvaldo Jimenez from the Estación Experimental Agropecuaria 

del INTA “Las Breñas”. 

 

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