بررسی باکتری‌های تجزیه کننده تانن جداسازی شده از پوست پسته و مدفوع بز تغذیه شده با پوست پسته

Introduction

By- products of Pistachio contain high amount of tannin. Every year, tons of Pistachio by- products are produced in many parts of Iran (about 450000 tons per year) (1). Due to containing high amount of available carbohydrate and proteins, soft hulls of pistachio (P- SH) can be used as an energy source in ruminant's nutrition. However, the presence of high tannin content is the preliminary limitation of using this by- product as a ruminant feed (2 & 3).

Tannins are toxic, high molecular weight, astringent, bitter plant secondary hydrolysable and condensed polyphenols that either bind and precipitate or condense proteins (4). The toxicity of phenolic compounds in the environment has encouraged studies of bacteria that are able to tolerate and/or metabolize high levels of these compounds (1 & 5). Besides altering taste and palatability of diets, tannins can reduce feed intake (6 & 7). Tannins also suppress rumen fermentation (8) and produce an insoluble protein- tannin complex that is poorly digested in the rumen and lower digestive tract, which inhibits microbial enzymes involved in fiber degradation (9).

Low levels of tannins (<5% dry weight) can protect protein from bacterial deamination (7 & 10), and prevent bloat (11). Ruminal microbial population, a cocktail of bacteria, protozoa, and fungi are not yet fully investigated in the presence of tannin. So, decreasing tannins effect would allow several feed trees to be included into different ruminant farming systems, which could improve the nutritional status and productivity of animals besides allowing a more environmentally friendly ruminant production (12).

Different groups of bacteria, fungus and yeasts with the ability of tannin reduction by producing tannase have been isolated (5, 13- 14). Among these, bacteria capable of degrading or tolerating tannins have been isolated from the alimentary tracts of several animals, e.g., koalas (Phascolarctos cinereus) (15), goats (Capra hircus) (5), horses (Equus caballus) and sheep (Ovis aries) (5). Also, different strains of tannin degrading bacteria were isolated from feces samples of ruminants which feed with tannin rich plants (16).

The aim of this study was to isolate and characterize tannin- tolerant bacteria from soft hulls of pistachio and the feces of goat before and after feeding on P- SH as tannin rich diet.

Materials and methods

Sample collection and bacterial enumeration: Fresh pistachios were purchased from the market and the soft hulls were collected for microbiological examinations such as enumeration of heterotrophic and tannic acid degrading bacterial (TDB) population as well as isolation and characterization. Two male goats maintained at the Agricultural Sciences Research Farm of the Islamic Azad University, Khorasgan- Isfahan branch in Isfahan Province, were used in this study. From September to October, goats were offered the same diets (Table 1). They were fed twice each day at 8: 00 AM and 4: 00 PM while they had free access to water. Fecal samples were collected with fecal bags before and after feeding. The samples suspended in sterilized phosphate buffer saline (3) and carefully mixed with a homogenizer and a vortex test- tube mixer in the biotechnology laboratory of Islamic Azad University Isfahan- Khorasgan branch.

Table 1- Goat’s diet in adaptability and experimental period.

*The concentrate includes: 24% barn, 47% barley, 24% corn and a 5% mixture of vitamins and mineral salts. Adaptability and Experimental periods were done for one and three weeks respectively.

Enumeration of heterotrophic and tannic acid degrading bacterial (TDB) population: Total heterotrophic counts were done in triplicate on nutrient agar (NA) plates after preparation of serial dilutions (10^{- 1} to 10^{- 7}) of samples. Plates were incubated for 3 d at 30°C. Thereafter, the number of colony- forming units (CFUs) was counted. Tannic acid degrading bacterial (TDB) population was also enumerated by counting the CFU/mL using spread plate method on MSM agar supplemented with 0.5 g/l tannin after serial dilutions preparation (10^{- 1} to 10^{- 7}) of samples. After 3- 5 d of incubation at 37°C, the colonies were counted and colony forming unit (CFU/mL) was calculated.

Enrichment cultures and isolation of the TDB strains: Aliquots of 1.0 g of P- SH or 1ml of fecal suspensions were added to 50 ml of liquid minimal salts medium (MSM) containing as follows, and incubated without shaking at 30°C:

MSM composition per liter:

Na₂HPO₄- 2H₂O: 2.7 g; KH₂PO₄: 1.4 g; (NH₄) ₂SO₄: 0.5 g; MgSO₄- 7H2O: 0.2 g; yeast extract 0.02 g.

And 10 ml trace elements solution of the following final composition per liter:

Na₂EDTA- 2H₂O: 12.0g; NaOH: 2.0g; MnSO₄- 4H₂O: 0.4g; ZnSO₄- 7H₂O: 0.4g; H₂SO₄: 0.5 ml; Na₂SO₄: 10.0 g; Na₂MoO₄- 2H₂O: 0.1 g; FeSO₄- 7H₂O: 2.0 g; CuSO₄- 5H₂O: 0.1 g; CaCl₂: 1.0 g. Tannic acid was supplied as the sole carbon and energy source, at concentrations of 0.5 g/l (17).

After three weeks with one further transfer, enrichment cultures showing turbidity were selected for isolation experiments. The isolation and purification was also performed on solid MSM plates supplemented with 2 g/l tannic acid. Bacterial isolates were then characterized according to Bergey’s manual of Systematic Bacteriology (18), by Gram staining and biochemical tests. Isolated strains were stored as liquid culture containing 30% sterile glycerol (v/v) in - 80°C.

The maximum tolerable concentration (MTC) of tannins was selected as the highest concentration of tannic acid that allows growth after 24- 48h. The increasing concentration of tannic acid (1, 2, 4, 8, 16, 24, 32, 64 g/l) on MSM agar plates were used for testing the MTCs (19).

Biodegradation experiments: After purification of colonies the degradation ability of each pure colony was confirmed by cultivating on tannin- treated MSM agarmedia and incubation at 37°C for 24- 48 hours. After incubation, bacterial growth and tannin hydrolyzing ability were confirmed by observing clear zones around the colonies. For biodegradation experiments, in the media supplemented with 15 g/l of tannic acid, tannin concentration were measured by bovine serum albumin (BSA) precipitation assay after 24, 48 and 72 hours of incubation and the biodegradation percentage was calculated. For this purpose, the bacterial suspensions were centrifuged (3, 000 g at 4°C for 10 min) and the supernatant was taken to determine tannins. For each sample, 1mL of a 1 mg BSA/mL acetate buffer stock solution was combined with 1ml of tannic acid solution and precipitation reactions were allowed to proceed under refrigeration for 18 hr after which samples were centrifuged (3, 000 g for 10 min) and the precipitate was dissolved in SDS solution (1% w/v). The aliquot of 1 mL of resulted solution was combined with 3ml TEA- SDS solution (7%TEA & 1% SDS). After several minutes 1mL of FeCl₃ was added and the optical density was measured after 60 min incubation at room temperature, then absorbance was measured at 520nm, zeroing the spectrophotometer with a tube containing all of the reagents plus water in place of the extract. Tannin contents were determined according to calibration curve (R²=0.971) which was constructed from commercial tannic acid ranging from 0- 20 g/l.

Statistical analysis: All data was analyzed using the Statistical Analysis System (2001). Multiple comparisons of means were done with the Duncan test method (at Sig<0.05).

Results

Heterotrophic, Tannin- degrading bacteria (TDB) of P- SH and TDB population of goat feces before and after feeding on tannin rich diet were estimated. According to Fig. 1A, heterotrophic bacteria of P- SH were estimated about 310×10⁵ CFU/gr. It was also cleared that after feeding on tannin rich diet TDB population of goat feces increased significantly (Fig. 1B). Tannin degradation ability of isolates was confirmed by the formation of clear zone around the colonies on tannin- treated MSM agarmedia. According to our results, 54% of tannin degrading bacteria were isolated from goat feces after feeding on tannin rich diet (Fig. 2A), 69% of them were Gram negative bacilli while only 31% was Gram positive bacilli belonging to the bacillus genera (Fig. 2B). Based on the morphology, carbohydrate utilization capacity and biochemical tests, they were identified as Klebsiella, Pseudomonas, Bacillus, Escherichia and Enterobacter species (Fig. 3B). Among isolated bacteria 71.4% of them could tolerate the high concentration of 64 g/l of tannin in their media while only 7.2% of them were able to tolerate the maximum tannin concentration of 16 g/l (Fig. 2C).

Tannin concentrations in the media were measured by BSA precipitation assay after 24, 48 and 72 hours of incubation and the biodegradation percentage was calculated. According to results; bacterial isolates of goat feces degraded tannin more than 72% after 72 hours of incubation so that isolates 1, 2 and 3 could degrade tannic acid (15 g/l) about 99% after 3 days of incubation. In the case of P- SH isolates, the biodegradation percentage was between 17- 75% (Fig. 2).

Fig. 1- Heterotrophic and Tannin degrading bacteria (TDB) of P- SH (A).TDB population of goat feces before and after feeding on tannin rich diet (B).

Fig. 2- Percentage of isolated bacteria according to source of isolation (A), identified genus (B) and Maximum Tolerable concentration of Tannin (C).

Fig. 3- Biodegradation percentages of tannic acid (15 g/l) after 24, 48 and 72 h of incubation; (1- 7: bacterial isolates from goat feces after feeding on tannin rich diet, 8, 9: before feeding on tannin rich diet and 10- 13: bacterial isolates from P- SH).