INTRODUCTION:

Nowadays, there is an increased public awareness about the risk of developing cross-resistance of pathogens to antibiotics. There have been numerous reports regarding the occurrence of foodborne diseases caused by pathogenic microorganisms such as Escherichia coli O157: H7, Campylobacter and Salmonella (Wendel, 2009; D Mello, 2004, Mermin and Griffin 1999). There are a number of consumer have worry related with use of antibiotics in feed animals, included contamination residue of poultry products and antibiotic resistant microorganisms pathogens. These affairs have resulted in commendation to minimize the use of antibiotics as growth promoters in animals feed. Researchers focus on natural material that have positive impact on animal health and production like probiotic and prebiotic, synbiotic, essential oil, herbal and organic acids (Al-Kassie et.al. 2008ab, Jameel, 2017, Jameel, 2018, Abdaljaleel et.al., 2018; Ali et.al., 2018).

Organic systems were not permitted to use antibiotics, on the contrary conventional production. Thus, both conventional and organic poultry production need alternative methods to improve growth and performance of poultry (Diaz-Sanchez et al., 2015). Feed industry and the food production strip yet suffer from losses due to the contamination of feed with pathogenic microorganism and their related impacts in the animal and human healthy. Humans utilized organic acids as natural food preservatives and hygiene supportive with, regard to the bacterial growth, boost freshness and shelf life of eaten food items (Islam et al., 2008 and Waseem et al., 2016).

Organic acids are chemical weak acids, which prohibit or completely detain the propagation and colonization of pathogenic microorganisms in the GIT which, made from it suitable replacement to antibiotic growth promoter in poultry. Thus, reducing the rivalry for the nutrients as well as production of hurtful microbial metabolites (Theron, 2010).

Organic acids are weak acids, which means that a certain proportion of the molecules remain undissociated, depending on the acid’s pKa value and the ambient pH level. Organic acids have been ability to penetrate the cellular membrane of the bacteria in the undissociated form because they are lipid soluble. (Young and Foegeding, 1993). Once inside the cell, the acid releases protons in the more alkaline environment, resulting in the reduction of intracellular pH proton (H+) (Baronofsky et al., 1984, Biggs and Parsons, 2007). This influences microbial metabolism, inhibiting the action of important microbial enzymes (Warth, 1991). The bacterial cell is forced to expend energy to expel the protons, leading to an intracellular accumulation of acid anions, depending on the pH gradient across the membrane, The RCOO- anions produced from the acid can interrupt DNA and protein synthesis. (Nursey, 1997). This condition is unfavorable for enter pathogenic bacteria colonization, such as E. coli, Campylobacter, and Salmonellas (Humphrey and Lanning, 1988, Dibner and Buttin, 2002).

MATERIAL AND METHODS:

Normal saline:

This solution was prepared by dissolving 8.5 grams of sodium chloride in one litter of distilled water (Cowan and Steel, 1985). The solution was distribute into test tubes as 5 ml volumes and sterilized by autoclave at 121ºC for 15 minutes.

McFarland Standard:

McFarland Standard is a chemical solution of barium chloride (BaCl₂₎ and sulfuric acid (H₂SO₄. It is used to calibrate the convergent number of microorganism in a liquid suspension by analogy the turbidity of the test suspension. The accuracy checked by using a spectrophotometer with a 1-cm light path a 0.5 McFarland Standard has an absorbance reading of 0.08 to 0.1 at 625-nm. (McFarland, 1907).

Procedures:

The E. coli O157:H7 suspension was prepared from the pure culture and inoculating in a suitable broth, then compared the turbidity visually of the test suspension with the McFarland standard (McFarland, 1907).

Preparation of media:

Tryptic Soy Agar (TSA, pH7, Difco), Tryptic Soy Broth (TSB, pH7, Difco) and sorbitol MacConkey agar (SMA) (pH7, Oxoid) were prepared as accord by the manufacturers. TSB was dole out into either test tubes (10ml) or 250 ml Erlenmeyer ﬂasks (100ml) and sterilized by heating at 121°C for 15min.

Preparation of inocula:

Bacteria were maintained on TSA (pH7·2) slants at 5°C and activated by transferring loop inocula into 10ml TSB and incubating at 37°C. Three consecutive 24 h loop transfers were made before inoculating into ﬂasks containing 100ml TSB or TSBG. Cultures incubated at 37°C for 18h were used in subsequent acid challenge studies.

Feed acidification:

Fife hundred (gm) of feed was covered by aluminum foil and sterilization by autoclave at 121ºC for 15 minutes then added different concentration of acetic, citric acid and blend together (0.5, 1, 1.5) % respectively.

Feed contamination:

The feed sample inoculated with 5 McFarland standard of E. coli O157:H7 and incubated for 2- 4 hours after that cultured on sorbitol maconcky agar the growth is assessed after incubation for a defined period of time for (18–24 h).

Determination of pH:

The pH values of samples were determined with a pH meter probe (Model 430; Corning Inc.) during the time. A maximum of 20 milliliters of distilled water can be added to 100 grams of the sample without changing the pH of the product (Bennett and Luft, 1959).

Procedures for determining growth or survival time:

The animal feed sample was sterilized using autoclave and subsequently added organic acids in the following (0.5, 1 and1.5) percentage respectively, pH was recorded and then log 5 of McFarland of E. coli O157:H7 cfu/ml was inoculate then incubated for 24 hours. The bacterial count was done every 60 Min. E. coli O157:H7 were serially diluted in sterile 0·1% peptone water and agar plate in duplicate (0·1ml) on TSA Plates were incubated at 37°C for at least 48h before colonies were counted. Two or three replicate tests were carried out.

Bacterial enumeration:

Aliquots (1 ml) of sample were tenfold serially diluted with 9 ml of sterile buffered peptone water and 0·1 ml of sample or diluent was spread plated on to Sorbitol MacConkey agar (Difco). All samples were incubated at 37°C for 24 h, and then colonies were enumerated.

Result:

Table (1) gives the parameters estimates for the logistic regression model with significant (p˂0.05) effect of different percentage of acetic acid, citric acid and blind (0.5, 1, and 1.5) on growth of E. coli O157: H7 compared with control group. The influence of the acid concentration was appeared in the table (1) when compared between the different groups, the blend acids (B31.5%) groups (3.88±0.041) showed high significant effect (p˂0.05) than acetic and citric acid group (A, C) groups at 60 minutes of time.

Table (1): Influence of different percentage of acetic, citric and blind (0.5, 1, and 1.5) on viable counts of the E. coli O157H7 mean ± SE.

E. coli O157:H7 Count (cfu/ml)						Groups
180 minutes	120 minutes	60 minutes	0 time	pH	No. of sample	Groups
8.58±0.120 ^a	7.25±0.044^a	5.76±0.053^a	5.16±0.030^a	7.00±0.10	4	Co-
0.00±0.000 ^b	1.51±0.066^b	4.29±0.041	4.52±0.078	4.66±0.03	4	A1
0.00±0.000^b	1.17±0.022	4.20±0.108	4.45±0.075	4.22±0.03	4	A2
0.00±0.000^b	0.70±0.056	3.97±0.053	4.29±0.041	3.91±0.01	4	A3
0.00±0.000^b	1.66±0.080^b	4.30±0.042	4.55±0.099	4.53±0.02	4	C1
0.00±0.000^b	1.19±0.026	4.22±0.033	4.34±0.049	4.20±0.03	4	C2
0.00±0.000^b	0.61±0.069	3.97±0.036	4.30±0.030	3.87±0.01	4	C3
0.00±0.000^b	1.37±0.075^d	4.08±0.048	4.38±0.068	4.37±0.05	4	B1
0.00±0.000^b	0.35±0.035	3.99±0.048	4.26±0.046	4.14±0.02	4	B2
0.00±0.000^b	.00±0.0000	3.88±0.041	4.19±0.035	3.83±0.02	4	B3

- Small letters bearing variable subscripts are significantly differences among group

-(p˂0.05)

The available growth time of E. coli O157H7 that contaminated chicken feed acetic, citric acid and both (blend) of them (0.5, 1, 1.5) % on the growth of E. coli O157:H7 were showed exhibited tolerance for 120 minutes except the blend group (B3) 1.5%, was showed no growth (0.00) in all group after 180 minutes of incubation.

The figures (1, 2) showed that the effect of different concentrations of acetic and citric acid (0.5, 1, and 1.5) on the E. coli O157:H7 load. The growth of E. coli O157:H7 were observed at 0, 60, 120, 180 minutes. The bacterial load was highest at 0 minutes, while lowest on 180 minutes post treatment for all three concentrations of both the acids. The growth regression with the time and the viability lost after 180 minutes of incubation at 37 ˚c where no growth observed and there is no significant effect between the influences of acetic and citric acid on the viability growth of E.coli O157H7.

Figure (1) effect of different level of acetic acid (0.5, 1, and 1.5) on growth viability of the E. coli O157H7.

Figure (2) effect of different level of citric acid (0.5, 1, and 1.5) on growth curve of the E. coli O157H7.

In the figures (3) showed that the curve line of growth regression with time and the viability lost after 180 minutes of incubation at 37˚c where no growth observed, except (B3) treatment showed the stopped of growth at 120 minutes of incubation at 37 ˚c. The figures appears there were a significant effect (P < 0.05) on the growth curve line compared with A, C groups.

Figure (3) effect of different level of blend acetic and citric acid (0.5, 1, and 1.5) on growth curve of the E. coli O157H7.

DISCUSSION:

Weak organic acids (acetic, citric acid) can control growth of pathogenic bacteria (Davidson, 2002). The antimicrobial effect of organic acids is due to the ability of undissociated molecules can diffuse across bacterial cell membranes and ionize to release protons in the cytoplasm, thereby depressing intracellular pH and inhibiting the nutrient transport system and enzymatic reactions (Cherrington, 1991and Booth et al., 1989). Other factors affecting acid inhibition of microbial pathogens include: specific effects of the acid or acid anion on cellular enzymes or membranes, the internal pH values and buffering capacity of cells, proton pumping at the expense of cellular ATP, and facilitated transport of acid molecules (Beales, 2004, Booth et al., 1989, Warnecke et al., 2005)

The table (2) showed the significant (P > 0.05) effect among the (A, C, B) groups and (CO) group, so the group B3 (0.5, 1, 1.5) percentage showed the highest effect among groups (4.19±0.03, 3.88±0.04, 00±0.00) at all-time measuring. Previous studies have shown the effects of organic acids in killing E. coli O157:H7. The various effect of different amount of acetic, citric acid and both (blend) of them (0.5,1,1.5) % on the growth of E. coli were observed at table (2) and figure (1,2,3) with time 0, 60, 120 and 180 minute. The bacterial load was highest at 0 minute, while lowest on 180-minute post treatment for all three concentrations. Acetic, citric acid and both reduced the E. coli load in 60,120, incubation time. Result revealed that blend (B) groups was more effective than acetic and citric (A, C) in all (0.5, 1.1.5) percentage concentration. The result is consistent with what the researcher (Amer, 2017) report that the viable E. coli O157:H7 (5 log cfu/ml), when incubated at 37 ⁰C in acidified nutrient will decline the growth and no growth was observed after 120 minutes. Organic acid stress should induce membrane structure changes in E. coli O157:H7 to prevent inﬂux of undissociated acid molecules (Sinensky, 1974), the change of the membrane structure dependent on both pH and organic acid(Yuk & Marshall, 2005). The antimicrobial effect of organic acid on E. coli O157:H7 has been demonstrated by several studies (Chikthimmah, et al., 2003, Comes and Beelman, 2002, Kondo, et al., 2006). Organic acid recognized as safe (GRAS) for their ability to kill E. coli O157:H7, including acetic, citric, lactic, malic, and fumaric acids. (Buchanan et al., 1996).

Figure (1) showed there were a significant effect (p>0.05) of acetic acid in all concentration (0.5, 1, 1.5) on the number, E. coli O157:H7 (5.76±0.053, 4.29±0.041, 4.20±0.108) log cfu/ml, respectively with incubation times of up to 60 min. In the incubation times up than 120 min by 1.66±0.080, 1.19±0.026, 0.61±0.069 respectively compared with control group.

The influence of citric acid against E. coli O157:H7 was represented in Figures (2). The results showed that all reducing numbers of E. coli O157:H7 4.55±0.099, 4.34±0.049, 4.30±0.030 respectively with incubation times of up to 60, and by 1.51±0.066, 1.17±0.022, 0.70±0.056 log cfu/ml respectively with incubation times of up to 120 min. However, this result agrees with (Itelima, 2014) who, that noted at pH levels of 4.6, 3.8 and 3.0, citric acid reduced counts of E. coli O157:H7 (p>0.05) with increase in time for up to 40min compared with control group.

The result showed in Figure (3) thus, at pH levels of 4.37, 4.14, 3.83, blend (acetic and citric acid) was highly significant reduce the counts by 4.08±0.048, 3.99±0.048, 3.88±0.041 log cfu/ ml respectively with incubation time of up to 60 min, and by 1.37±0.075, 0.35±0.035, 0.00±0.00 respectively with incubation times of up to 120 min compared with control group.

Akyurek et al. (2011) reported that broiler chickens fed diets containing organic acid blends had less pathogenic bacterial loads such as coliforms and Clostridia but greater beneficial bacteria such as Lactobacilli in the ileum compared with those fed diets containing AGPs. The antibacterial activity increases with decreasing pH value (Russell, 1992). The lower pH conditions thus protect the animal from infection especially at young ages.

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Received on 25.10.2018 Modified on 14.11.2018

Research J. Pharm. and Tech. 2019; 12(5):2468-2472.

DOI: 10.5958/0974-360X.2019.00414.1