Introduction

Clostridium perfringens (C. perfringens) is a gram-positive, anaerobic, and rod-shaped bacterium that survives longer than vegetative cells, such as coliforms (e.g., Escherichia coli and Enterococci) (Gerba, 2015). This bacterium is distributed in nature and could be found in the intestine of animals and humans (Taghi Akhi et al., 2015). The ingested C. perfringens can produce enterotoxin in the intestine, which is capable of binding to the epithelial cells of the intestine leading to damaged cell membranes of the host. As a result, glucose absorption is prevented, while secretion of sodium and chloride increases due to altered permeability. In animal nutrition, reduced nutrient uptake causes a decline in feed efficiency, and removing C. perfringens from the intestines could improve growth performance (Zaffarano, 2003).

Feeding antibiotics for improving livestock performance has been associated with antibiotic resistance concerns. Resistant bacteria will be transported from animals to humans through food consumption (Zaffarano, 2003). Foodborne illness is the central problem of pathogenic resistant bacteria (Chan et al., 2018). Antibiotic-resistant C. perfringens strains are becoming a significant health concern due to their role in bacterial foodborne illnesses. However, increasing concern about antibiotic resistance is forcing farmers to find alternatives (Modi et al., 2014). These alternatives include probiotics and prebiotics, which can prevent the disease and improve growth characteristics. To alter the intestinal microbiota, the consumption of prebiotic carbohydrates like resistant starch (RS) is recommended (Herrmann et al., 2017). This type of carbohydrate is not digested in the upper gastrointestinal tract. According to Liu et al. (2020), Dietary fiber isolated from sweet potato residues, as a type of RS, significantly decreases the concentrations of C. perfringens.

On the other hand, the food industry has an ever-growing interest in using natural antimicrobials due to the health risk of chemical additives, which can improve food stability and safety against pathogens (Santas et al., 2010). Some chemicals, including phenolic compounds, which are a significant group of biologically active chemicals found in some foods, plants, and residual plants are generally recognized as safe (GRAS) (Lambert et al., 2001) and are often used as natural preservatives in food. These compounds are utilized as natural antimicrobials and have a great potential for controlling the growth of pathogens (Cetin-Karaca and Newman, 2015). In addition to their antimicrobial activity, they are of particular interest as natural alternatives to synthetic preservatives in food (Bouarab-Chibane et al., 2019).

Moreover, phenolic compounds and flavonoids are synthesized by many plants and fruit species that are utilized in traditional medicine or diets (Tungmunnithum et al., 2018). Kim et al. (2011) reported the antimicrobial activity of some plant-derived phenolic compounds. In a study by Jianu et al. (2012), the thymol derived from dill seeds had a strong antimicrobial impact on C. perfringens. However, there are no available reports about the synchronic effect of phenolic compounds and prebiotics on C. perfringens as a pathogenic bacterium. Therefore, this investigation was carried out to evaluate the antibacterial effect of the phenolic compound of the extracts of grape pomace, pistachio peel, and pomegranate pomace on C. perfringens in the presence or absence of RS.

Materials and Methods

 Providing Material and Preparing Extracts

Pistachio peel (from Nut and Pistachio Peel Commerce Co., Mashhad), Pomegranate pomace (from Naariran Co., Saveh), and grape pomace (from SunSunShahd Co., Urmia) were purchased. The RS (Fibersol2) was purchased from Karen Nutrilife Co., Yazd, Iran. For preparing the extracts, 50 g of air-dried and powdered (0.5 mm) pomegranate pomace, grape pomace, and pistachio peel were extracted separately with 300 mL methanol 99.5% and were kept and shaken every 30 min at room temperature for 30-32 h. Afterwards, the extracts were filtered through Whatman 42 mm and located in a water bath under sterile air condition. Finally, the extracts were collected and weighted after combining and evaporating all methanolic fractions.

Determining the Total Phenolic Compounds and Total Tannin Content

The Folin-Ciocalteu and standard tannic acid method were used to determine the total phenol and tannin content (Makkar, 2000). Briefly, tannins containing extracts were transferred into the test tube at three different quantities of 0.02, 0.05, and 0.1 ml. Next, 1.25 ml sodium carbonate solution and 0.25 ml Folin-Ciocalteu reagent were added and vortexed well. Absorbance was recorded at 725 nm after keeping at room temperature for 40 min. The total phenols were measured based on a standard calibration curve and expressed on a dry matter basis. Afterwards, the tannins were removed from the extract. For this aim,100 mg Polyvinylpyrrolidone (PVPP) was poured into the test tube, 1 ml distilled water was added, vortexed well, and kept at 4ºC for 15 min. The test tube was centrifuged at 3000 rpm for 10 min and the supernatant was collected. The phenolic content of this supernatant was calculated according to the Folin-Ciocalteu method. This non-tannin compound was expressed based on dry matter. After calculating total phenolic and non-tannin compounds, the result of subtracting the non-tannin from the total phenolic was considered as total tannin.

Determining Minimum Inhibitory Concentration

The minimum inhibitory concentration (MIC) of the extracts with or without RS was determined according to the recommendation of the National Committee for Clinical Laboratory Standards (NCCLS, 2000) using the broth microdilution method (96-well plate) in duplicates. Briefly, 0.02 g of each extract and 0.02 g of RS were added separately to a 2 mL sterile brain heart infusion (BHI) broth medium and were vortexed well to reach a final concentration of 10⁴ ppm as the stock solution. Two-fold dilutions were prepared to obtain the concentrations of 50, 100, 200, 400, 800, 1600, and 3200 ppm of each extract in 2 mL BHI broth + dimethyl sulfoxide. The standard strain of C. perfringensATTC 13124was cultivated on Luria-Bertani (LB) broth to activate the bacteria (Sigma-Aldrich, Germany). Afterwards, the bacterial suspension was prepared in turbidity equal to 0.5 McFarland standard tubes (5×10⁵ CFU/mL). Then, 200 µl of each dilution of grape pomace extract, pistachio peel extract, pomegranate pomace extract, grape pomace extract+RS, pistachio peel extract+RS, pomegranate pomace extract+RS with 6 µL of bacterial suspension of C. perfringenswas added to each well. Finally, the plate was incubated at 37ºC for 24 h in anaerobic conditions. After the incubation period, an enzyme-linked immunosorbent assay microplate reader was applied to measure the absorbance of each well at 630 nm (BIOTEK ELX 800, USA). The MIC was the lowest concentration of extracts with or without RS that prevented the visible growth of bacteria (Andrews, 2001).

Statistical Analysis

The data were recorded at 0 and 24 h (i.e., the times of inoculation and after incubation) and analyzed by the t-test (P≤0.05) using the SAS software version 9.1 4 (Statistical Analysis Systems, Cary, NC, USA) for determining the difference between the growth rates of bacteria at two-hour intervals.

Results

The total phenolic compounds of grape pomace, pomegranate pomace, and pistachio peel extracts were 2.7%, 14.94%, and 11.74% of dry matter, respectively. The total phenolic compounds of pomegranate pomace and grape pomace were the highest and lowest, respectively. The tannin contents of grape pomace, pomegranate pomace, and pistachio peel extracts were 2.167%, 3.634%, and 1.906% of dry matter, respectively. Therefore, the highest tannin content was observed in pomegranate pomace.

The MICs of the extracts of grape pomace, pomegranate pomace, and pistachio peel for C. perfringens are shown in Table 1. According to this table, C. perfringens could grow in a culture media containing diverse dilutions of grape pomace extract, except 800 ppm. As shown in Table 1, C. perfringens did not grow in 100 and 200 ppm of pomegranate pomace extract. In contrast, the MIC of pistachio peel extract showed that 100 ppm of pistachio peel extract could inhibit the growth of C. perfringens.

Table 1. The Minimum Inhibitory Concentration results of grape pomace extract, pomegranate pomace extract, and pistachio peel extract for Clostridium perfringens

*: Significant difference in bacterial growth between 0h and 24h (P≤0.05)

NS: Not Significant difference in bacterial growth between 0h and 24h (P>0.05)

The MICs of RS are presented in Table 2 which indicates that RScould not inhibit the growth of C. perfringens in all dilutions. As shown in Table 3, RS + grape pomace extract prevented C. perfringens growth in 400, 800, 1600, and 3200 ppm dilutions. Therefore, the MIC of RS + grape pomace extract for C. perfringens was 400 ppm dilution. The MICs of RS + pomegranate pomace extract for C. perfringens revealed that the combination of RS and pomegranate pomace extract could not inhibit C. perfringens growth. However, the dilutions of 50 and 100 ppm of RS + pistachio peel extract could restrain its growth (Table 4).

Table 2. The Minimum Inhibitory Concentration results of Resistant Starch for Clostridium perfringens

*: Significant difference in bacterial growth between 0h and 24h (P≤0.05)

NS: Not Significant difference in bacterial growth between 0h and 24h (P>0.05)

Table 3. The Minimum Inhibitory Concentration results of grape pomace extract, pomegranate pomace extract, and pistachio peel extract + Resistant Starch for Clostridium perfringens

*: Significant difference in bacterial growth between 0h and 24h (P≤0.05)

NS: Not Significant difference in bacterial growth between 0h and 24h (P>0.05)

Table 4. Growth of Clostridium perfringens in different dilutions of phenolic extract ±Resistant Starch (brief)

+: bacterial growth

- : Lack of bacterial growth

Discussion