حداقل غلظت بازدارندگی عصاره فنولی و نشاسته مقاوم بر کلستریدیوم پرفرینجنس: مطالعه آزمایشگاهی

نوع مقاله : تغذیه- بهداشت

نویسندگان

1 گروه علوم دامی، دانشکده کشاورزی و منابع طبیعی، دانشگاه محقق اردبیلی، اردبیل، ایران

2 گروه علوم دامی و صنایع غذایی، دانشکده دامپزشکی، دانشگاه سمنان، سمنان، ایران

3 گروه پاتوبیولوژی، دانشکده دامپزشکی، دانشگاه سمنان، سمنان، ایران

چکیده

< p >زمینه مطالعه:  نقش کلستریدیم پرفرینجنس در ایجاد بیماری‌هایی که از طریق خوراک منتقل می‌شوند، مسئله مهمی است که در صنعت خوراک انسان و دام وجود دارد. افزایش نگرانی‌ها دربارۀ مقاومت آنتی‌ بیوتیکی انسان را وادار به یافتن جایگزین‌هایی برای آنتی‌بیوتیک‌ها کرده است.
هدف: هدف از تحقیق حاضر بررسی فعالیت ضد میکروبی عصارۀ تفاله انگور، عصارۀ پوستۀ پسته و عصارۀ تفالۀ انار در حضور یا عدم حضور نشاسته مقاوم به‌عنوان پری بیوتیک بر کلستریدیوم پرفرینجنس بود.  
روش کار: برای این هدف، نشاسته مقاوم (فایبرسول 2) فراهم شده و عصارۀ تفالۀ انار، پوستۀ پسته و تفالۀ انار آماده شد. میزان فنول کل و تانن عصاره‌ها به ترتیب به‌وسیله روش فولین سیوکالتو و استاندارد اسید تانیک تعیین شد. فعالیت ضد میکروبی عصاره‌ها در ترکیب یا بدون نشاسته مقاوم با استفاده از روش حداقل غلظت بازدارندگی علیه باکتری کلستریدیوم پرفرینجنس ارزیابی شد.
نتایج: نتایج نشان داد که 100 پی‌پی‌ام از عصارۀ پوستۀ پسته توانست به‌عنوان یک بازدارندۀ رشد برای کلستریدیوم پرفرینجنس عمل کند. نشاسته مقاوم به تنهایی قادر به ممانعت از رشد کلستریدیوم پرفرینجنس نبود. درحالی‌که 400 پی‌پی‌ام از مخلوط عصارۀ تفالۀ انگور و نشاستۀ مقاوم از رشد کلستریدیوم پرفرینجنس ممانعت کرد؛ در مقابل، عصارۀ پوستۀ انار در هر دو حالت بدون نشاسته مقاوم و در ترکیب با نشاسته مقاوم مانع رشد این باکتری نشد. از سویی دیگر، عصارۀ پوستۀ پسته در مخلوط با نشاسته مقاوم و بدون نشاسته مقاوم در مقایسه با سایر تیمارها نتوانست از رشد باکتری کلستریدیوم پرفرینجنس جلوگیری کند.
نتیجه‌گیری نهایی: عصارۀ تفالۀ انگور در هر دو حالت، همراه با نشاسته مقاوم و بدون نشاسته مقاوم، توانست در ممانعت از رشد کلستریدیوم پرفرینجنس موثر باشد.

کلیدواژه‌ها


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

 

Dilution

Maen-0h

Mean-24h

f-value

v. equal   test

T-value

significant

Grape   Pomace Extract

50

0.087

0.087

<.0001

Unequal

1.0000

NS

100

0.1215

0.159

0.1003

Equal

0.1880

NS

200

0.122

0.156

0.1409

Equal

0.0642

NS

400

0.174

0.2515

0.2966

Equal

0.1250

NS

800

0.2785

0.338

0.7487

Equal

0.0222

*

1600

0.443

0.526

0.8705

Equal

0.1400

NS

3200

0.8

0.9185

0.8562

Equal

0.0754

NS

Pomegranate   Pomace Extract

50

0.102

0.223

0.1209

Equal

0.0291

*

100

0.1075

0.228

0.0883

Equal

0.0792

NS

200

0.1295

0.311

0.3349

Equal

0.0792

NS

400

0.1755

0.3705

0.6289

Equal

0.0014

*

800

0.4165

0.6015

0.4097

Equal

<.0001

*

1600

0.3725

0.569

0.3711

Equal

0.0007

*

3200

1.0035

1.167

0.6962

Equal

0.0067

*

Pistachio   Peel Extract

50

0.112

0.117

0.7487

Equal

0.2999

NS

100

0.128

0.1435

0.5903

Equal

0.0052

*

200

0.2055

0.2975

0.3390

Equal

0.0038

*

400

0.2975

0.357

0.9252

Equal

0.0101

*

800

0.503

0.8455

0.6500

Equal

0.0017

*

1600

0.903

1.4465

0.5096

Equal

0.0007

*

3200

1.384

1.927

0.4320

Equal

0.0098

*

*: 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

Dilution

Maen-0h

Mean-24h

f-value

v. equal   test

T-value

Significant

50

0.088

0.3675

0.9023

Equal

0.0003

*

100

0.083

0.388

0.1688

Equal

0.0025

*

200

0.087

0.369

<.0001

Unequal

0.0045

*

400

0.0865

0.3685

1.0000

Equal

<.0001

*

800

0.08

0.362

0.4097

Equal

0.0005

*

1600

0.0775

0.3795

0.2513

Equal

<.0001

*

3200

0.0775

0.3675

0.1228

Equal

0.0029

*

*: 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

                       
  

 

  
  

Dilution

  
  

Maen-0h

  
  

Mean-24h

  
  

f-value

  
  

v. equal    test

  
  

T-value

  
  

Significant

  

grape   pomace extract + Resistant Starch

50

0.08

0.4165

0.1335

Equal

0.0072

*

100

0.086

0.336

0.0606

Equal

0.0070

*

200

0.0995

0.1795

0.1651

Equal

0.0204

*

400

0.1275

0.173

0.1666

Equal

0.1409

NS

800

0.17

0.2135

<.0001

Unequal

0.0656

NS

1600

0.291

0.3355

0.1491

Equal

0.0351

NS

 

3200

0.415

0.4605

0.4568

Equal

0.0087

NS

pomegranate   pomace extract + Resistant Starch

50

0.0915

0.2495

0.1295

Equal

0.0234

*

100

0.104

0.2445

0.0688

Equal

0.0169

*

200

0.118

0.25

0.1521

Equal

0.0345

*

400

0.151

0.329

0.5325

Equal

0.0030

*

800

0.221

0.3885

0.6067

Equal

0.0107

*

1600

0.3335

0.499

0.9252

Equal

0.0117

*

3200

0.5295

0.735

0.3753

Equal

0.0197

*

pistachio   peel extract + Resistant Starch

50

0.0965

0.095

0.8591

Equal

0.6855

NS

100

0.1145

0.12

0.8591

Equal

0.2280

NS

200

0.151

0.1745

0.5903

Equal

0.0023

*

400

0.2135

0.2655

0.6199

Equal

0.0325

*

800

0.3435

0.5445

0.4097

Equal

0.0030

*

1600

0.5895

0.9885

1.0000

Equal

0.0003

*

3200

0.942

1.456

0.3119

Equal

<.0001

*

*: 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)

 

Dilution

50

100

200

400

800

1600

3200

grape   pomace extract

-

-

-

-

+

-

-

pomegranate   pomace extract

+

-

-

+

+

+

+

pistachio   peel extract

+

+

+

+

+

+

+

RS

+

+

+

+

+

+

+

grape   pomace extract + RS

+

+

+

-

-

-

-

pomegranate   pomace extract + RS

+

+

+

+

+

+

+

pistachio   peel extract + RS

-

-

+

+

+

+

+

+: bacterial growth

- : Lack of bacterial growth

 

Discussion

 

The structure of polyphenol, the microorganism strain, and the evaluated dosage are some factors that affect bacterial metabolism and growth (Hervert-Hernandez and Goni, 2011). The outer membrane of gram-negative bacteria is a lipopolysaccharide membrane (Kalambheet al., 2017). Consequently, gram-positive bacteria are more sensitive to polyphenols due to their wall composition (Ghimire et al., 2017).

Recent findings demonstrated that phenolic compounds may bind to bacterial cell membranes and disturb their function leading to the inhibition of cell growth (Kemperman et al., 2010). Singh et al. (2019) argued that polyphenols generate hydrogen peroxide and can alter the microbial membrane permeability. In addition, polyphenols can bind bacterial cell membranes and alter membrane function resulting in prevention from their growth (Singh et al., 2019).

Bouarab-Chibane et al.(2019) noticed that hydrogen bonding of hydroxyl groups of polyphenols (e.g., catechins and theaflavins) to lipid bilayers of cell membrane controls the antimicrobial mechanism of polyphenols. The configuration of these polyphenols is influenced by molecular structure at the time of binding to the bilayer surface, and they form hydrogen bonds with the lipid head groups. Selma et al. (2009) reported that the main genera involved in the metabolism of many phenolics (e.g., isoflavones, flavonols, flavones, and flavan-3-ols) are C. and Eubacterium.

Dolara et al. (2005) found a shift in fecal bacterial composition from Bacteroides, Clostridium, and Propionibacterium spp. to Bacteroides, Lactobacillus, and Bifidobacterium spp. in rats that consumed proanthocyanidin-rich grape extract. Larrosa et al. (2009) and Tzounis et al. (2008) stated that the growth of some Bifidobacteria and Lactobacilli were stimulated or remained comparatively unaltered by phenolic compounds, such as resveratrol. However, the growth of C. perfringens was inhibited by catechin and epicatechin, as the types of polyphenols. Yamakoshi et al. (2001) evaluated the growth inhibitory activity of grape seed extract against C. perfringens. They stated that the growth of C. perfringens was not prevented by the phenolic extract.

Bouarab-Chibane et al. (2019) stated that the phenolic compounds of plant extracts are natural alternatives to synthetic preservatives in food. Li et al. (2015) reported that pomegranate extract increased the growth of Lactobacilliand bifidobacteria. On the other hand, it inhibited the growth of the Bacteroides fragilis group, Clostridia, and Enterobacteriaceae in stool cultures. In another study carried out by Rosas-Burgos et al. (2016), the most sensitive strains to the constituents of pomegranate by-products were gram-positive intestinal pathogenic species, such as C. perfringens. Naziri et al. (2012) demonstrated that the different antibacterial activities of the methanolic extract of pomegranate peel may be due to the variations in the antibacterial substances, namely tannins and phenolic substances. Kavak et al. (2010) investigated Pistacia terebinthus extract, as a potential antioxidant, antimicrobial, and possible β-glucuronidase inhibitor. These authors concluded that Pistacia terebinthus leaf extract had antimicrobial activity against Staphylococcus aureus as a gram-positive bacteria, while it did not have sufficient antimicrobial activity against E. coli.

Tzounis et al. (2011) suggested that phenolic compounds (flavan-3-ol monomers) may influence the bacterial population of the large intestine even in the presence of carbohydrates and proteins. It appears that polyphenols have a prebiotic effect on the modulation of gut microbiota and exert antimicrobial activities against pathogenic gastrointestinal bacteria (Kawabata et al., 2019). As mentioned before, the host physiology is dependent on gut microbiota (Umu et al., 2013), and the distinct physicochemical and metabolic properties of fibers result in a different impact on community composition from the ingestion of dietary (Umu et al., 2015). Therefore, the prebiotic characteristics of RS may be due to the non-digestibility of carbohydrate fractions for colonic bacteria that influence the host gut health (Spencer, 2011). The weight of the total gastrointestinal tract increased by RS consumption in the animals. Elevated bacterial mass, fermentation end-products (Slavin, 2013), and augmented metabolically active tissue in the colon (Souza da Silva et al., 2014) result from the mentioned effect of RS. The prebiotics selectively stimulate the growth of beneficial bacteria, such as Lactobacilli and bifidobacteria (Samal et al., 2015), while suppressing the growth of toxogenic and proteolytic bacteria, including C. perfringens, Streptococcus spp., and Staphylococcus spp. (Samarasinghe et al., 2003; Rohin et al., 2014). We found that although RS did not affect growth prevention of C. perfringens, grape pomace extract and RS had a synchronic inhibitory effect on this strain.

Conclusion

We concluded that the grape pomace extract prevented C. perfringens growth. The RS had no inhibitory effect on the growth of this bacterium. However, the treatments of RS  pomegranate pomace extract and RS + pistachio peel extract could not inhibit the growth of C. perfringens. On the other hand, the RS + grape pomace extract could well suppress the growth of C. perfringens by a synchronic inhibitory effect.

Acknowledgments

The authors of this article express their appreciation to the faculty of Veterinary Medicine, department of Pathobiology in the Semnan University, for their help in this research.

Conflict of Interest

The authors declared no conflict of interest.

 

 

 

 

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