The effect of shallot (Allium ascalonicum L.) by-product as an antibacterial and alternative phytobiotic on characteristics of small intestine of broiler

S Mozin^{1, 2}, D Rosyidi², O Sjofjan² and E Widodo²

¹ Faculty of Animal Husbandry and Fishery, University of Tadulako, Palu, Central Sulawesi, Indonesia
selvymozin@yahoo.com
² Faculty of Animal Husbandry, University of Brawijaya, Jalan Veteran, Malang, Indonesia

Abstract

Shallot (Allium ascalonicum L.) by-product has antibacterial properties due to the greater presence of an active substance (quercetin) in this part of the shallot than in edible parts. The first phase of the study aimed to determine the inhibitory effect of shallot by-product meal and juice extract on the growth of the bacteria  Lactobacillus sp, E. coli and Salmonella sp. A nested factorial design was used with the factors of physical form of shallot by-products (meal and juice) and the level of shallot by-products extract (juice of 1.5%, 3%, 4.5%; and meal 1%, 2%, 3%). Bidistilled water was used as a negative control and tetracycline 30 µgml^-1 as a positive control.

Extracts of shallot by-product meal were more effective as antibacterial agent than those of shallot by-product juice extract, whereas inhibitory effect was found more effective on Gram negative bacteria than the Gram positive bacteria. The objective of second phase of the study was to investigate the characteristics and the numbers of bacteria (Lactobacillus sp,  E. coli and Salmonella sp) in the intestinal digesta of chickens kept for 42 days. Starter and finisher diets were offered ad-libitum. Collections of small intestines and their digesta were done at the end of the study. A completely randomized design was used in the second phase to determine the effect of treatments on villus height, crypt depth, the ratio of villus height and crypt depth, pH and  viscosity of the digesta and the number of bacteria Lactobacillus sp,  E. coli and  Salmonella sp in the intestinal digesta. The treatments used were the level of shallot by-product meal, namely: T0 (0%), T1 (0.5%), T2 (1%), T3 (1.5%) and T4 (2%). Shallot by-product meal had no negative effect on the characteristics of small intestine and the numbers of three groups of bacteria in the digesta. However, further study is needed  to characterize the quantity and quality of its active substance. Thus, it can be applied as an alternative phytobiotic and useful to reduce the environmental problems.

Keywords: extract, feed additive, gut microbial, poultry, quercetin

Introduction

Fried shallot is one of the products at the city of Palu, Central Sulawesi, Indonesia. The product becomes specific because the raw material used was shallot (Allium ascalonicum L.) which only grows in Palu city, Sigi and Donggala regency.  In the process of manufacturing fried shallot, shallot by-product was generated. The by-product has not been widely utilized so that this can pollute the environment. Percentage of shallot by-product generated due to the process of fried shallot production was about 17%. Accordingly, an increase in the production of fried shallot in Palu can increase  production of shallot by-product.  Roldán et al (2008) stated that the onion (Allium cepa L.) industry produced by-product about 15% of the total production, and they increased every year. Shallot by-product consists of dry skin, outer and inner layers,  and top-bottom of the shallot. Dry skin onion (Allium cepa L.) contains high concentrations of quercetin aglycone and calcium, while the top-bottom had a high mineral content (Benítez et al 2011). He stated the outer skin can be used as a source of flavonols, with good antioxidant activity and relatively high levels of dietary fiber.  Total isolated phenols and quercetin of onion skin reported by Skerget et al (2009) were three to five times more than the edible part of the onion. Distribution of quercetin on each layer of the Blonska onion according to Wiczkowski et al (2003) was varied, where the deeper layers produced the smaller amounts of quercetin (layers I - VIII generated as much as 53.5%; 16.5%; 12.2%; 10, 9%; 4.6%; 1.7%; 0.4% and 0.2% respectively).

Shallot (Allium ascalonicum L.) had an antimicrobial property and can widely inhibit pathogenic and non-pathogenic bacteria (Amin and Kapadnis 2005). As an antimicrobial, shallot has the property to control microorganisms in the gut of poultry. It is thought to control gut microorganisms, promoting the performance of the host and contributing to maintain the health of the host. Shallot by-product like other Allium species might be used as a phytobiotic in poultry feed. The aim of the present study was to investigate theanti bacterial activities of extracted shallot by-product meal and juice extract and the effect of shallot by-product meal on the characteristics of small intestine and total bacteria in ileal digesta.

Materials and methods

Experiment 1 (an in vitro study)

Extraction of shallot by-product meal and juice

Shallot by-product was obtained from home industry of fried shallot at Palu city. This by-product was washed and sun-dried. The dried by-product was then finely ground to form shallot by-product meal. Shallot by-product juice was produced by blending shallot by-product with the addition of water at a ratio of 1 : 2 (water: shallot by-product). Shallot by-product meal and juice were then extracted by maceration. Extraction process was performed by mixing 100 g of sample with 200 ml of 98% ethanol (Safithri et al 2011) with a slight modification using an orbital shaker (Genhart) for 3 days shaking. The extract was then filtered with sterile Whatman paper No. 1. The filtrate was evaporated in a vacuum using a rotary evaporator (Eyela SB-110) and stored at 5°C in a refrigerator in the dark sample bottle.

Analysis of quercetin

Quercetin analysis was conducted by using the method of Phani et al (2010). Preparation of quercetin standard was done by dissolving 5 mg of quercetin standard in 10 ml of methanol. One gram of shallot meal and juice were then placed into a 10 ml volumetric flask and dissolved in 10 ml of methanol. The samples were then sonificated for 20 minutes and filtered with filter paper (0.45 µm). The process was carried out 3 times. A 50 ml of extract sample was re-filtered by using a 0.45 µm filter paper. The filtrate produced was injected into the HPLC DAD (Shimadzu) and measured at a wavelength of 355 nm.

Antibacterial testing

Antibacterial effects of extracted shallot by-product meal and juice were done by Agar Diffusion Method. Media MHA (BB) was used for culturing E. coli and Salmonella sp that were synchronized with a solution of 0.5 Mc Farland (Enyi-Idoh et al 2011) and MRSA (Oxoid) for Lactobacillus sp. These bacteria were obtained from chicken. The wells were made with a cork borer of 5 mm in the media that had been cultivated with bacteria and spilled with 50 µm extract of shallot by-product meal and juice according to the treatments. Bacteria were incubated at 37^oC for 24-48 hours and the inhibition zones were measured as the area of inhibition of bacteria.

Parameters and statistical analysis

Inhibition of the bacteria (Lactobacillus sp, E. coli and Salmonella sp) was the parameter measured by using a nested pattern of completely randomized design. The treatments of the study were physical form of shallot by-product (meal and juice) and the level of extracted shallot, namely: juice (1.5%; 3%; 4.5%) and meal (1%, 2%, and 3%). Bidistilled water was used as a negative control (M- and J-) and tetracycline 30 µgml^-1 as a positive control (M+ and J+). A total of 30 treatments were applied with 3 replications. Data were analyzed using Minitab 17 and the differences between treatments were tested using LSD test (Steel and Torrie 1991).

Experiment 2 (in vivo study)

Histopatology of small intestine

A total of 200 chickens were randomly allocated to 25 pens. The small intestine of chickens was collected after feeding with basal diet ad-libitum for 42 days (Table 1).  The treatments were the addition of shallot by-product meal level as phytobiotic level. For intestinal characteristics, the intestine from Meckel's diverticulum to ileocaecal junction with the length of 40 mm was placed into a 10% formalin solution. Intestinal samples were dehydrated using different concentration of alcohol and then were infiltrated with a solution of multilevel xilol (Tissue Tex Processor) for 90 minutes. Furthermore, samples were put in paraffin and sliced as thick as 3-5 µm using a microtome. Samples were then placed on glass objects and stained with haemotoxylin and eosin (HE). Samples were dehydrated again using multi-level alcohol, and then clarified using xylol for 60 minutes. The samples were allowed to dry at room temperature and prepared for observation (Saki et al 2012). Each sample was measured for villi height and crypt depth counted using a microscope (Olympus BX51) and camera (Olympus XC10), viewed using OlyVIA software. The intestinal digesta for viscosity and pH measurement was collected by using a syringe and saline solution. The digest was mixed with distilled water at a ratio of 1: 1 (Piel et al 2005 with slight modification) and the mixture was centrifuged at a speed of 3,500 g for 10 minutes. Supernatant obtained was measured with a viscometer (Brookfield Viscometer FF-LV) at 30 rpm and a pH meter (WTW pH 3110). 

The number of Lactobacillus sp, E. coli and Salmonella sp of the digesta

Intestinal digesta samples of 42-day-old chickens were placed in a sterile bottle. The digesta was homogenized and diluted using 0.1% peptone solution. Selective media was used to grow each bacterium. MRS Agar (Oxoid) for Lactobacillus sp, VRBA (Oxoid) for E. coli and SSA (BB) for Salmonella sp. After incubation at 37^o C for 24 hours for E. coli and Salmonella sp and 48 hours for Lactobacillus sp, total bacteria was calculated using the Automatic colour colony counter version 5.0 (Interscience).

Parameters and statistical analysis

Parameters measured consisted of digesta pH and viscosity, total digest bacteria (Lactobacillus sp, E. coli and Salmonella sp), villi height, crypt depth and villi height ratio to crypt depth. The treatments tested were the addition of various levels of shallot by-product meal, namely: T0 (0%), T1 (0.5%), T2 (1%), T3 (1.5%) and T4 (2%). A completely randomized design was used in this study and data were analysed by using Minitab 17.

Results and discussion

Quantitative analysis of quercetin

Quercetin analysis of shallot by-product by HPLC determined 0, 81 mg/g extract of shallot by-product meal (Figure 2) and 0,024 mg/g extract of shallot by-product juice (Figure 3).

Figure 1. HPLC chromatogram of standard quercetin	Figure 2. HPLC chromatogram of extract of shallot by-product meal
Figure 3. HPLC chromatogram of extract of shallot by-product juice

Antibacterial activity

The inhibition capacity of extracted shallot by-product against Lactobacillus sp, E. coli and Salmonella sp (Table 2) was improved with the increase in concentration of extracted of shallot by-product meal and juice extracts.

Although, the shallot by-product juice extract could inhibit the growth of all three types of bacteria, the inhibition capacity was lower than the extracted shallot by-product in the form of meal. This is probably caused by concentration of the active substance (quercetin) present in shallot by-product meal being higher than in juice. According to Cowan (1999), antibacterial mechanism of quercetin might be through membrane disruption and inactivation of extracellular proteins of bacteria by forming an irreversible complex but the exact mechanism is still unclear. In addition, the inhibitory activity of quercetin is also caused by the inhibition of DNA gyrase (Plaper et al 2003). Quercetin binds the sub-unit gyrB of E. coli through DNA gyrase and inhibits the activity of the enzyme ATPase (Plaper et al 2003). According to Martini et al (2004) and Koo and Jeon (2009), quercetin has been investigated extensively through its bioactivity in inhibiting Gram positive and negative bacteria through inactivity of extracellular proteins. Other reports, on the other hand, indicated that quercetin had  no inhibitory activity against E. coli bacteria as found by Rauha et al (2000) with quercetin dose of 500 ug/ml. Souza et al (2010) used 30 mg/ml quercetin and found no effect no inhibitory activity to E. coli and Salmonella typhi. The inhibitory capacity of shallot by-product, either in form of meal or juice, was more powerful against the growth of Gram-negative bacteria than those of Gram-positive one. This might be due to the thicker peptidoglycan layer in Gram-positive bacterial cellwall (Wada et al 2012). It is stated that mechanically and chemically, the cell wall of peptidoglycan could protect bacterial cell.

Characteristics of small intestinal and the numbers of Lactobacillus sp, E. coli and Salmonella sp in intestinal digesta

Intestinal villi have a correlation with the capacity to absorb the chicken diet, the longer and wider the intestinal villi, the more efficient the nutrient absorption process. Digest pH has a relationship with the health of the gut, where pH value low indicates good conditions of the gut and thus subsequent improvement in the growth rate (Dono 2012). Feed additives stimulate the development of gut and nutrient utilization. One way of studying the effects of feed additives on the growth of the gut is through the morphology test. The lack of effect of treatment in this study indicated that the active substance present in shallot by-product could not stimulate the intestinal villi to optimally develop so that the increase in surface area for nutrient uptake is also not optimal. The ratio of the villi height and crypt depth according to Petrolli et al (2012) indicated the degree of maintenance of the intestinal villi in relation to the degree of urgency of the synthesis of enterocytes by crypt, so it is an indicator of intestinal health.

In an in-vitro study, shallot by-product meal and juice extract had antibacterial property then it was applied in an in-vivo study in broiler diet. According to Lan et al (2003) Lactobacillus sp and Enterococcus sp are the Lactic acid bacteria (LAB) found in chicken gastrointestinal. The active substance in shallot by-product tended to increase Lactobacillus sp bacteria and to decrease the number of E. coli and Salmonella sp in the gut. Hence, this by-product is relatively useful in maintaining the balance between non-pathogenic and pathogenic bacteria. Villus height and crypt depth increased relatively due to the addition of shallot by-product. The reason of this is not known. Quercetin as an active substance present in the shallot by-product may play a role to this phenomenon.

Conclusion

• Extract of shallot by-product meal produced more inhibitory capacity on Lactobacillus sp, E. coli and Salmonella sp than the extract of shallot by-product juice, and the more the concentration of the extract the more powerful the inhibitory capacity.

  
  

• There was no negative effect of the use of shallot by-product meal on the characteristics of small  intestine  and the numbers of Lactobacillus sp, E. coli and Salmonella sp in the intestinal digesta of broiler chickens.

Acknowledgements

This article is part of the research for doctoral degree in  Doctoral Program of Animal Science, Post graduate Program, Faculty of Animal Husbandry, University of  Brawijaya, Indonesia.  The author would acknowledges the Directorate General of  Higher Education, Ministry of  Research Technology and Higher Education,  Republic of Indonesia  for financial support  for this research.

References

Amin M and Kapadnis B P 2005: Heat Stable antimicrobial activity of Allium ascalonicum against bacteria and fungi. Indian Journal of Experimental Biology, v.43, n.8, p. 751-754.

Benítez V, Mollá E, María A, Martín-Cabrejas, Aguilera Y, López-Andréu F J, Cools K, Terry L A and Esteban R M 2011 : Characterization of industrial onion wastes (Allium cepa L.): dietary fibre and bioactive compounds. Plant Foods for Human Nutrition, v. 66, issue 1, p. 48–57.

Cowan M M 1999: Plant products as antimicrobial agents. Clinical Microbiology Reviews, v.12, n.4, p.564-582.

Dono N D 2012: Nutritional strategies to improve enteric health and growth performance of poultry in the post antibiotic era. PhD Thesis. The College of Medical, Veterinary and Life Sciences, University of Glasgow.

Enyi-Idoh, Hovana K, James U S, James E, Egbobor E M, Egba A G, Eta E S and Nwakaku O A 2011 : Antibacterial and phytochemical studies of Allium sativum. New York Science Journal, v.4, n.4, Article#21 p.123-128 Retrieved April 2011, from http://www.sciencepub.net/newyork/ny0404/

Koo H and Jeon J G 2009 : Naturally occurring molecules as alternative therapeutic agents against cariogenic biofilms. Advances in Dental Research, v.21, n.1, p.63–68.

Lan P T N, Binh LT and Benno Y 2003 : Impact of two probiotic Lactobacillus strains feeding on fecal lactobacilli and weight gains in chicken. Journal of General and Applied Microbiology, Japan, v.49, p.29-36.

Martini N D, Katerere D R P and Eloff J N 2004 : Biological activity of five antibacterial flavonoids from Cambroteum erythrophyllum (Combretaceae). Journal Etnopharmacol, v.93, issue 2-3, p.207-212.

Petrolli T G, Albino L F T, Rostagno H S, Gomes P C, Tavernari F C and Balbino E M 2012 : Herbal extracts in diets for broilers. Revista Brasileria de Zootecnica,v.41, n.7, p.1683-1690.

Phani Ch R S, Vinaykumar Ch, Umamaheswara rao K and Sindhuja G 2010: Quantitative analysis of quercetin in natural sources by RP-HPLC. International Journal of Research in Pharmaceutical and Biomedical Science, v.1, issue 1, Article#5, p.19-22 Retrieved July 2010, from http://www.ijrpbsonline.com/archives1.html

Piel C, Montagne L, Sėve B and Lallės J 2005: Increasing digesta viscosity using carboxymethyl cellulose in weaned piglets stimulates ileal goblet cell numbers and maturation. Journal of Nutrition, v.135, n.1, p. 86-91.

Plaper A, Golob M, Hafner I, Oblak M, Solmajer T and Jerala R 2003: Characterization of quercetin binding site on DNA gyrase. Biochemical and Biophysical Reserach Communication, v. 306, n.2, p.530–536.

Rauha J, Remesa S, Heinonenb M, Hopiab A, Kähkönen M, Kujalac T, Pihlajac K, Vuorelaa H and Vuorela P 2000 : Antimicrobial effects of finnish plant extracts containing flavonoids and other phenolic compounds. International Journal of Food Microbiology, v.56 issue 1, p.3–12.

Roldán E, Sánchez-Moreno C, Begoňa de Ancos and Cano M P 2008: Characterisation of onion (Allium cepa L.) by-products as food ingredients with antioxidant and antibrowning properties. Food Chemistry, v.108, issue 3, p.907–916.

Safithri M, Bintang M and Poeloengan M 2011 : Antibacterial activity of garlic extract against some patogenic animal bacteria. Media Peternakan, Indonesia, v.34, n.3, p. 155-158 Retrieved December 2011 from http://medpet.journal.ipb.ac.id/index.php/mediapeternakan/article/view/3945

Saki A S, Harcini R N, Rahmatnejad E and Salary J 2012 : Herbal additives and organic acids as antibiotic alternatives in broiler chickens diet for organic production. African Journal of Biotechnology, v.11, n.8, p.2139-2145.

Ṥkerget M, Majhenič L, Bezjak M and Knez Ẑ 2009: Antioxidant, radical scavenging and antimicrobial activities of Red Onion (Allium cepa L.) skin and edible part extracts. Chemical and Biochemical Engineering Quarterly, v.23, n.4, p.435-444.

Souza L D, Wahidulla S and Devi P 2010: Antibacterial Phenolics from the Mangrove Lumnitzera racemosa. Indian Journal of Marine Sciences, New Delhi, v.39, n.2, p. 294-298 Retrieved June 2010, from http://nopr.niscair.res.in/handle/123456789/10027

Steel R G D and Torrie J H 1991 Principle and Procedure Statistics 2^nd Edition. McGraw-Hill Book Co, Inc, Singapore.

Wada A, Kono M, Kawauchi S, Takagi Y, Morikawa T and Funakoshi K 2012 : Rapid discrimination of gram-positive and gram- negative bacteria in liquid samples by using NaOH sodium dodecyl sulfate solution and flow cytometry. Plos One, v.7, Issue 10. Retrieved October 2012, from http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0047093

Wiczkowski W, Nèmeth K, Buciñski A and Piskula M K 2003 : Bioavailability of quercetin from flesh scales and dry skin onion in rats. Polish Journal of Food and Nutrition Sciences, v.12/53, n.1s Special issue, p. 95–99.

Received 20 January 2015; Accepted 18 March 2015; Published 1 April 2015

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