Quantitative Determination of Tannin Content in Some Sorghum Cultivars and Evaluation of its Antimicrobial Activity
Abdel Moneim E. Sulieman ,
Fatima M. Issa
Elamin A. Elkhalifa
In the present study, tannin content was investigated in three Sudanese sorghum cultivars. In addition, the inhibitory effect of natural tannins isolated from sorghum grains as well as that of commercial tannins was detected against Escherichia coli, Staphylococcus aureus, Salmonella typhimurium (bacteria), Aspergillus niger, Aspergillus flavous (mould) and Saccharumyces cerivisae (yeast). The results indicated that natural tannin from sorghum has a notable antimicrobial activity against most of the examined microorganisms, the higher antimicrobial activity among all examined organisms was found against Salmonella typhymurium and Saccharomyces cerivisae. The results also indicated that commercial tannins are more effective than natural tannins. This study has shown the importance of commercial tannins and sorghum tannins as antimicrobial and preservative agents.
Sorghum is not only a staple cereal for millions of poor farmers in the world, but it has also good feed and forage value. Sorghum grain is used in the manufacture of different items such as wax, starch, alcohol, dextrose sugar and edible oil.
In Sudan sorghum is the most important cereal crop in terms of acreage and production and a human food. Sorghum improvement in Sudan started in 1918 (Mahmoud, 1978). A good collection of local and introduced sorghum types were made and studied by Evelyn in the early forties. Since early sixties and up to date a lot of work has been done on genetic improvement of sorghum and production of Tabat and Dabar sorghum cultivars are good examples of this improvement.
Tannins are phenolic compounds that precipitate protein. They are composed of a very diverse group of oligomers and polymers (Palo, 1985; Waghorn et al., 1990). The presence and consequent interaction of tannins and proteins in the seeds of cereals and legumes have been believed to be of the factors involved in reduced protein digestibility (Bressani et al., 1983; Gupta, 1987). Most berries, such as cranberries and blueberries contain both hydrolysable and condensed tannins (Vattem et al., 2005; Puupponen-Pimiä et al., 2001).
Tannins are important ingredient in the process of tannin leather. Oak bark has traditionally been the primary source of tannery tannin, though synthetic tanning are also in use today.
The objectives of the present study were to quantify tannin content in some sorghum grains samples as well as detection of its antimicrobial activity.
MATERIALS AND METHODS
The Plant Material
Sorghum seeds samples from three cultivars of sorghum were used in the present
study; these cultivars included Tabat, Dabar and Feterita.
The first two cultivars were obtained from Wad- medani local market, while the
third cultivar was obtained from Gedarif local market (May, 2006). All grain
samples were freed from foreign materials like stones, sand and dust. The seeds
were then washed with water, dried and milled into fine flour using a laboratory
mill (Christ and Norris Limited, England).
Determination of Tannins Content
The tannin contents were determined using Folin Denis Reagent as described by
Makkar et al. (1993). In that method, a standard calibration curve was
prepared and the Absorbance (A) against concentration of tannins at specific
wave length was estimated as follows:
Suitable aliquots of the tannin-containing extract (initially: 0.05, 0.2 and 0.5 mL) were pipetted in test tubes, the volume was made up to 1.00 mL with distilled water, then 2.5 mL of sodium carbonate reagent were added. Then the tubes were shaken and the absorbance was recorded at 725 nm after 40 min. The amount of total phenols was calculated as tannic acid equivalent from the standard curve.
Testing of Tannic Acid and Natural Tannins for Antibacterial Activity
Tannic acid at 0.5, 10. 20, 2.5 and 25 g L-1 concentrations was
dissolved in distilled water and tested against Staphylococcus aureus
(ATCC 25923), Esherichia coli (ATCC 25922), Salmonella typhimurum
(ATCC 14028) (bacteria)and mould (Aspergillus niger and Aspergillus
flavous-locally isolated strains) and one species of yeast (Saccharomyces
cervisae-commercial strain). The examined microorganisms were obtained from
the Medical laboratory-University of Gezira. The examined microorganisms were
thoroughly mixed with 200 mL of sterile molten nutrient agar, which was maintained
at 45°C. Twenty milliliter aliquots of the inoculated agar were distributed
into sterile Petri dishes. The agar was left to set and each of these plates
were cut using a sterile cork borer and the agar discs were removed. Alternate
cups were filled with 0.1 mL sample of tannic acid, extracted tannins using
adjustable pipette and allowed to diffuse at room temp for 2 h. The plates were
then incubated in the up right position at 37°C for 10 h; 2 replicates were
carried out for the tannic acid, extracted tannins against each of tested organisms.
After incubation, the diameter of the resultant growth inhibition zones was
measured; averaged and mean values were tabulated as described by Bary et
al. (1970) and Cruickshank et al. (1975).
The results were interpreted in term of the commonly used terms (sensitive) and (resistant). Concentration of the tannic acid used was 10-20 g L-1 dissolved in sterile distilled water. Concentration of extracted tannins used were (1:10) dissolved in methanol.
Testing of Tannic Acid and Natural Tannins for Antifungal Activity
For antifungal activity the same method for bacteria was adopted using sabouraud
dextrose agar instead of nutrient agar. The fungal culture was maintained on
sabouraud dextrose agar and incubated at 25°C for seven days. The fungal
growth was harvested and washed with sterile distilled water and finally suspended
on 100 mL sterile distilled water and the suspension was stored in a refrigerator
RESULTS AND DISCUSSION
Figure 1 shows the concentration of tannins in the different
sorghum samples. High levels of tannin were found in Dabar sorghum compared
with the other sorghum cultivars samples. However, these findings were in agreement
with those of Harris and Burns (1970) who indicated that the seed coat colour
of sorghum grain was associated with the tannin content; the brown varieties
contained more tannin. In contrast, Bullard et al. (1980) found insignificant
correlation between tannin content and seed coat colour. While, Blession et
al. (1963) indicated the contribution of non-tannin compounds to the pigmentation
of the seed coat. On the other hand, Feterita sorghum also contained
high concentration of tannin but less than that of Tabat.
Antibacterial Activity of Tannic Acid and Natural Tannins
Tannic acid and natural tannins showed the higher antibacterial activity
against Salmonella typhimurium, (Table 1 and 2),
the inhibition zone diameters were 30, 25 mm and 29, 20 mm at the higher and
the lower concentrations, respectively. The result agreed with that reported
by Chung et al. (1993) who stated that Salmonella typhimurium
was sensitive to tannic acid, with antimicrobial activity at 10 to 20 g L-1
of tannic acid.
Tannic acid and natural tannins also showed antibacterial activity of 29-25
mm and 20-15 mm, respectively, against Escherichia coli and this result
agreed with that of Irobi et al. (1994).
||Level of Tannin content in sorghum cultivars
||The antifungal activity of natural tannins
|*Tested organisms, As.n = Aspergillus niger, As.f.
= Aspergillus flavous, Sa.c = Saccharomyces cerivisae
||The antifungal activity of tannic acid against examined organisms
(moulds and yeast)
|*Tested organisms, As.n = Aspergillus niger, As.f.
= Aspergillus flaveous, Sa.c = Saccharomyces cerivisae
Hara and Ishigama (1989), who observed inhibition of E. coli by extracted tannins from plants. Also Chung et al. (1993) demonstrated that tannic acid and propyl gallate inhibited the growth of food borne bacteria, including E. coli and Salmonella enterititis.
Tannic acid and natural tannins showed a higher antibacterial activity against Staphylococcus aureus, the inhibition zone diameters were 25, 30 mm and 20, 15 mm at the higher and the lower concentrations, respectively. Those results agreed with that reported by Hada et al. (1989) and Sakanaka et al. (1989). They stated that Staphylococcus aureus were inhibited by condensed tannins and the growth of various diarrhea-causing pathogens was inhibited by tea extracts.
Antifungal Activity of Tannic Acid and Natural Tannins from Sorghum
The study indicated that tannic acid and natural tannins caused complete growth inhibition of Aspergillus niger and Aspergillus flavous (Table 3 and 4). Hitokoto et al. (1980) studied the effect of 13 herbal drugs and 7 commercial dry condiments on growth and toxin production by several toxigenic Apergillus species and found that powdered cinnamon was the most effective inhibitor. Hitokoto et al. (1980) reported that thyme caused 10-90% inhibition of the growth of three toxingenic Aspergilli, but showed nearly complete inhibition (86-100%) of their toxin production.
The data also indicated that tannic acid and natural tannins caused inhibition growth of Saccharomyces cerivisae type of yeast, when the inhibition zones were 25, 30 mm and 20, 25 mm at the higher and the lower concentrations, respectively. The result agreed to that reported by Mullins and Nesmith (1988) who reported that Saccharomyces cerevisiae was inhibited by sorghum grains by its tannins. Other authors demonstrated that tannic acid inhibited the growth of various yeast species some species were inhibited by tannins at 25 g L-1, while others were inhibited at much higher concentration of tannins (125 g L-1).
The objective of this research was to determine tannin content in sorghum cultivars, variation in the amount of tannin in different sorghum cultivars was found. The cultivar Tabat had the lowest tannin content than Feterita and Dabar samples. The microbiological analysis indicated that commercial tannins have high antimicrobial activity against many harmful microorganisms, especially Salmonella typhimurium. However, the inhibitory effect increased with an increase in inhibitor concentration.
More studies are needed to determine the antimicrobial activity of natural tannins from sorghum grains. Economic studies are needed to assess the extraction of tannin from sorghum grains and also the usage of sorghum tannins in food processing.
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