Contents of tannins of cultivars of sorghum cultivated in Brazil, as determined by four quantification methods

Highlights

• Different methods of sorghum tannin quantification provide widely distinct results.

• A test of validation provides support for the generalized use of the method vanillin/HCl.

• Sorghum cultivars analyzed have wide diversity of tannin contents.

• Contents of sorghum tannins cannot be predicted by grain color.

Abstract

High tannin content in sorghum grains is an undesirable characteristic for poultry and pig feeding and represents a challenge for breeding programs. On the other hand, moderate content of tannins in sorghum may be beneficial in human diets because they exert anti-cancer, anti-inflammatory and reduced carbohydrate uptake effects, among others. The aim of this study was to compare tannin contents of twenty sorghum cultivars available in Brazil, as well as to compare results obtained with four methods of tannin quantification: butanol/HCl, vanillin/HCl, BSA/FeCl₃ and PVPP/Folin-Ciocalteu. The results obtained with butanol/HCl and vanillin/HCl were higher than with BSA/FeCl₃ and PVPP/Folin-Ciocalteu. A known amount of purified quebracho tannin was used to test the validation of methods of tannin quantification and vanillin/HCl stood out for its high accuracy degree. The sampling used reveals wide genetic diversity regarding tannin contents. The expectation of predicting tannin contents on basis of grain color seems unfeasible.

Introduction

Sorghum (Sorghum bicolor (L.) Moench) is the world fifth most important cereal crop cultivated, with an annual grain production around 5.3 MT (FAOSTAT, 2020). Sorghum is a staple crop in Africa, Central America and South Asia. There is a worldwide growing market of sorghum for use as animal feed. The nutritional and functional properties, as well as health benefits of sorghum have been reviewed (Xiong et al., 2019). Nutritionally, sorghum grain contains 4.4–21.1% protein, 2.1–7.6% fat, 1.0–3.4% crude fiber, 57.0–80.6% total carbohydrates, 55.6–75.2% starch and 1.3–3.5% total minerals (ash). In addition, sorghum contains phenolic antioxidants and represents an optional cereal for people with celiac disease (Ratnavathi & Komala, 2016).

Sorghum grains are protected by a broad diversity of phenolic substances, such as condensed tannins, 3–deoxyanthocyanidins, flavonoids and phenolic acids (Taylor and Awika, 2017, Xiong et al., 2019). Among cereals, sorghum cultivars stand out for containing tannins in the pigmented testa, some of them at expressive amounts, as much as 50 mg g⁻¹ or even more (Dykes and Rooney, 2006, Xiong et al., 2019). Sorghum grains are characterized by the presence or not of a pigmented testa and their relative content of phenols and tannins. Such phenotypic characteristics are controlled by genes B₁ and B₂ and the spreader gene S. Sorghum cultivars with no pigmented testa usually have low levels of phenols and no tannins. Other cultivars may have a pigmented testa and tannins (Asquith et al., 1983, Dykes and Rooney, 2006). Sorghum grains with dark color pericarp produce trace amount of tannins, while the tannin contents in sorghum cultivars with purple testa are much lower than those with brown testa (Cheng et al., 2009). In addition, the presence of tannins in sorghum grains are controlled by different natural alleles of the gene Tan1 (Wu et al., 2012). Another gene affecting tannin contents in sorghum, Tan2, has been identified recently (Wu et al., 2019).

The relationship between sorghum grain color and tannin content is not simple. Pigments in both pericarp and testa have influence on grain color. While genes B₁, B₂ and S affect tannin content and the presence of a pigmented testa, pericarp pigmentation is controlled by genes other than B₁B₂S (Maxson et al., 1972a). The color of sorghum grains is evaluated colorimetrically or by specific data acquisition and image processing. Sorghum grain color is expressed by three characteristics: L* (lightness), a* (redness – from green to red) and b* (blueness – from blue to yellow). Boren & Waniska (1992) tested the correlation between lightness and tannin content. They concluded that seed and seed coat colors are poor or inadequate indicators of tannin content. On the other hand, Sedghi et al. (2012) found a strong correlation between lightness and tannin content, using artificial neural network models.

According to the respective tannin content, three groups of sorghum cultivars are recognized: a) group I: low level tannin, 0–1.8 mg catechin equivalents (CE) g⁻¹ dry mass (dm); b) group II: medium level tannin, 6.4–15.5 mg CE g⁻¹ dm; c) group III: high level tannin, 15.5–56.3 mg CE g⁻¹ dm (Dykes & Rooney, 2006). Tannin content is visually evaluated by the presence/absence of a pigmented testa, rather than pericarp color. Sorghum type I is free of tannins, although values of CE up to 2.5 mg g⁻¹ (0.25%) may be obtained by the method vanillin/HCl (Boren & Waniska, 1992). In these cases, if suitable blanks are used in the analysis, sorghums type I may be found to be tannin free.

Tannins are phenolic compounds with astringent taste and high molecular mass (300–3000 Da). The role of tannins as chemical barriers against pathogens and herbivores is widely recognized (Salminen & Karonen, 2011). Tannin content in sorghum grains is a subject of concern, because tannins reduce food digestibility and causes weight losses in poultry and pig livestock (Pan et al., 2018). This anti-nutritive effect derives from the ability of tannins to form cross-linkages with macromolecules, such as proteins and polysaccharides (Sieniawska & Baj, 2017). FAO/WHO recommends an upper limit of 0.5% of tannins for sorghum grains and 0.3% for sorghum flour (Codex, 1989). Currently, tannin-free sorghum cultivars comprise over 99% of sorghum crops grown in the United States. Distinct varieties of sorghum are grown in other parts of the world. For example, high tannin varieties are cultivated in Africa (Mabelebele et al., 2015). Regarding human health, it is believed that sorghum tannins may be beneficial. Molecular interaction of tannins with starch, proteins and enzymes, associated with their high antioxidant activity, decreases digestibility, reduces caloric intake, bringing about benefits toward obesity and type 2 diabetes, in addition to other health benefits, such as anti-cancer and anti-inflammatory. Another functional aspect of tannins is their high antioxidant activity. Probably, distinct degrees of tannin content in sorghum grains are more adequate for distinct purposes. For example, low tannin sorghum grains are ideal for animal feed and perhaps also for general human nutrition, whereas median or high tannin grains could be more appropriate for special human diets.

Attempts have been made to compare methods of determination of tannin contents in sorghum (Maxson and Rooney, 1972b, Earp et al., 1981). Widely different contents are obtained by analyses of the same plant material, depending on the method used. Butanol/HCl and vanillin/HCl colorimetric methods have been used. Butanol/HCl involves an oxidative depolymerization and release of monomeric units of catechin, which are converted into red anthocyanidins in hot acid media. Vanillin/HCl involves a condensation of vanillin with catechin in acid medium (Price et al., 1978). Both methods do not exclude low molecular mass phenols and are time-dependent (Waterman & Mole, 1994). Other methods are based on the capacity of tannins to bind to polymers of nitrogen-containing units. In Hagerman’s method (Hagerman, 1980), tannins are complexed with bovine serum albumin (BSA). Tannin molecules embedded in the precipitated BSA/tannin complex are released and quantified colorimetrically with ferric chloride (FeCl₃). Another method uses polyvinylpolypyrrolidone (PVPP) for tannin complexation/precipitation, instead of proteins. The colorimetric method Folin-Ciocalteu, used for quantification of total phenols, is used twice in this method: first, for evaluation of the content of total phenols in the tested solution; after the complexation of tannins with PVPP, another Folin-Ciocalteau reaction is done determine the remnant phenols in the solution (Makkar, 2003). Tannic acid is recommended as reference standard for both methods based on tannin complexation. Criticisms have been raised against Makkar’s method: Folin-Ciocalteu reagent is liable to cross reaction with several non-target substances, such as aldehydes, ketones, amines, amino acids, nucleotides, proteins, carbohydrates, thiols, and vitamins (Fernández & Medina, 2014). In addition, low molecular mass phenols can adhere to PVPP. Among the four commented methods, vanillin/HCl has been used more frequently, chiefly due to rapidity and sensitivity. On the other hand, Waterman & Mole (1994) recommended that several methods be used for determination of tannin content.

No specific standard for quantification of tannins in sorghum grains have been pointed out, although catechin has been used mostly. Another commercial tannin standard that has been used is the red quebracho tannin from Schinopsis balansae or S. lorentzii. However, quebracho tannin is highly impure, containing around 20% tannins mixed with low molecular mass phenols (e.g. gallic acid), sugars, lignin and inorganic substances (i. e. calcium salts) (Venter et al., 2012). In order to refine the methods of quantification analysis based on quebracho tannin, it is recommended to purify the product (Hagerman, 2002). According to Venter et al., (2012) purified quebracho tannin is composed of profisetinidins and procyanidins, substances formed by units of catechins combined by C4–C6 and C4–C8 links, respectively.

The aim of present study was to evaluate the contents of tannins in grains of twenty sorghum cultivars available for use in Brazil. For this purpose, it was planned to carry out analyses using two colorimetric methods based on catechin equivalents (butanol/HCl and vanillin/HCl) and two methods based on precipitation of tannins with nitrogen-containing polymers and based on tannic acid equivalents (BSA/FeCl₃ and PVPP/Folin–Ciocalteu). In addition, it was aimed also to test the reliability of methods of tannin quantification. For this purpose, it was carried out analyses of a known amount of purified quebracho tannin, using quantification methods based on distinct tannin properties.