Effectiveness of Bioactive Combinations of Several Plant Substances to Inhibit the Growth of Escherichia coli and Salmonella sp

Pasaribu T, Sinurat AP, Wina E, Purwadaria T, Haryati T, Susana IWR. 2018 Effectiveness of bioactive combinations of several plant substances to inhibit the growth of Escherichia coli and Salmonella sp.. JITV 23(3): 112-122. DOI: http://dx.doi.org/10.14334/jitv.v23i3.1851 The use of antibiotic growth promoters (AGP) has been banned as feed additives in many countries, therefore the alternatives need to be found. An in vitro experiment was conducted to study the potential of combination of some plant extract to inhibit growth of pathogen bateria that normally occur in the poultry gastro intestinal tract and in vivo studies to evaluate the population of E. coli in the ileum, the immune response and blood profile of chicken. The combination of three plants bioactives (liquid smoke of cashew shells of Anacardium occidentaleor CLS, Phyllanthus niruri L. extract (EM), and Synzygium aromaticum extract (EDC) were formulated and evaluated for its effectiveness to inhibit growth of Escherichia coli and Salmonella sp. in vitro.The mixtures (KE) were then made in 3 different concentrations, i.e. 100 % KE, 50% KE, and 25% KE and studied their effectiveness to inhibit growth of E. coli or Salmonella sp. using microplate reader method. In biological assay, the bioactive combination was at a concentration of 0.0625% CAM + 0.0625% EM + 0.0313% EDC. The treatment consisted of 8 types of rations, each of it 2 replications and each replication consisted of 5 DOC. At the end of the experiment (35 days), blood was taken from 2 chickens at each replication. The results showed that the higher the concentration of the bioactive combination (KE100) the higher the ability to inhibit the growth of E. coli or Salmonella sp. The combination of bioactive substances CAM, EM, and EDC more effectively than Zn-bacitracin antibiotics to inhibit the growth of E. coli and Salmonella sp. The optimum concentration of KE with the similar effectiveness as the AGP was 25%. It was concluded that the combination of CAM, EM, and EDC was able to inhibit the growth of E. coli and even capable to eliminate the presence of Salmonella sp. In the biological assay, a combination of CAM, EM, and EDC either extract or powder form, high dose, medium or low does not


INTRODUCTION
In general, antibiotic additives (AGPs / antibiotics growth promoters) are given to poultry by mixing them into feed or drinking water to increase production and prevent disease (Griggs & Jacob 2005).Giving AGP aims to minimize and even to reduce the population of pathogenic microbes in the intestine so that the nutrients contained in the gut are more dominantly absorbed.Pathogenic bacteria commonly found in the digest tracts of chickens and humans are Escherichia coli and Salmonella spp.In chickens, the bacteria are eliminated/killed by giving antibiotic supplementation so that the growth of livestock can increase up to 5-6% and the use of feed is more efficient up to 3-4% (Butaye et al. 2003).However, the use of antibiotics in livestock such as chickens can cause bacterial resistance to antibiotics.For example, Campylobacter and Salmonella against fluoroquinolone antibiotics and the third generation cephalosporins which is a constraint to the use of antibiotics in livestock (Noor & Poeloengan 2005).Many countries have banned the use of AGP, including Indonesia, as stated in Law No. 18 of 2009 juncto Law no. 14 of 2014.The ban on the use of antibiotics as a growth booster have been effective since January 1, 2018.Therefore, other materials such as AGP alternatives such as bioactive substances are needed.Several bioactive substances have been tested for their activity against pathogenic microbes in vivo (Lopez et al. 2012), but some are still in vitro test (Hoque et al. 2007).
Many local plants contain bioactive substances that function as antibacterial, antioxidant or antifungal such as cashew nut shells (Anacardium occidentale), meniran (Phyllanthus niruri L.), and clove leaf (Syzygium aromaticum).Several studies have been conducted for this purpose, such as the use of bioactive from Aloe vera, turmeric, Curcuma zanthorrhiza and Morinda citrifolia plants (Sinurat et al. 2004;Sinurat et al. 2009;Bintang et al. 2007).
Cashew (Anacardium occidentale) has pseudo-fruit waste and shell.When the shell extracted will be obtained liquid (oil) called biofat or Cashew Nut Shell liquid (CNSL).The waste of cashew nut shells is around 45-50%, with production of 137,496 tons in 2016, cashew shells is obtained around 61.8732 68.748 tons (SPI 2015-17).The bioindustry product of cashew nut shell consists of biofat, biosmoke and biochar (Saenab et al. 2016).Of these products, biofat (CNSL) has been widely studied, while biosmoke and biochar products from cashew shells have not been studied.Biosmoke (liquid smoke) is the result of decomposition process of cashew shells after extracted using hexane.The pyrolysis process is carried out with high heat without oxygen initiated by combustion and followed by total or partial oxidation of the main product (Bridgwater 2004).Liquid smoke from cashew shells can reduce the amount of E. coli in vitro (Sinurat et al. 2018), and suppress the growth of Candida utilis (Pasaribu et al. 2017).
Syzygium aromaticum (cloves) contain the active compounds of phenol (eugenol, flavonoid, hydroxybenzoic acid, hydroxycinnamic acid and hydroxyphenyl propene (Cortés-Rojas et al 2014;Perez-Jiménez et al. (2010).Eugenol has been shown to have antifungal, antiseptic and insect repellents.Eugenol inhibits the growth of fungus by damaging the walls and permeability of cells resulting in impaired growth (Putri 2002).Clove water extract can inhibit the growth of Escherichia coli, Staphylococcus aureus and Bacillus cereus (Cortés-Rojas et al 2014).Sinurat et al. (2018) have identified bioactive substances in 11 plants.Based on the results of the identification that liquid smoke from cashew nut shells (Anacardium occidentale) is potential as antibacterial, Phyllanthus niruri L.extract as an antioxidant, and clove leaf (Syzygium aromaticum) as an antifungal.With these three functions, it is hoped that the bioactive combination of the three plants has a higher inhibitory effect on the growth of E. coli and Salmonella sp.
This study aims to determine the inhibition power of the liquid smoke combination of cashew nut shells (Anacardium occidentale), Phyllanthus niruri L. extract and clove (Synzygium aromaticum) leaf extract on growth of Escherichia coli and Salmonella sp. in vitro as information for in vivo testing as a substitute for AGP in poultry.

Preparation of extract and liquid smoke
The making of Phyllanthus niruri L. or clove leaf extraction and preparation of cashew nut shells was done at the laboratory of Ciawi Bogor, Indonesian Research Institute for Animal Production.The preparation of meniran or clove leaf extraction was done by inserting 1 gram of powdered meniran or clove leaves into the test tube, added with 10 mL methanol 70%, then put into ultrasonic for 30 minutes.Furthermore, centrifuged for 15 minutes at 2500 rpm, the top layer is taken with a pipette, this part is called as an extract of P. niruri L. (EM) or extract Syzygium aromaticum leaf (EDC).The production process of cashew shell liquid smoke was done in a small industry of liquid smoke and charcoal manufacture in Cinangneng Village, Bogor.Preparation of liquid smoke using cashew shells that have been previously extracted with hexane solution as described by Saenab et al. (2016).

In vitro inhibitory test
Test of inhibition of Escherichia coli and Salmonella sp carried out in the Laboratory of Microbiology Indonesian Research Institute for Animal Production (IRIAP) Bogor.E. coli and Salmonella sp used are collections of IRIAP cultures.Extracts of bioactive substances that have been prepared combined and observed for its effectiveness.The test dose refers to the optimum dose of each extract performed in vitro in the previous study (Sinurat et al. 2018).The test was done by microplate reader method to study the inhibitory power of bioactive substance, by inoculating E. coli or Salmonella sp into microplate which has 96 wells.Microplate reader is a tool to perform analysis of active compounds or microorganisms quickly based on turbidity measured with optical density (OD) using a spectrophotometer.The common detection modes measured in this tool are the absorbance and intensity of fluorescence (Petersen et al. 2014).Resistance to the growth of E. coli and Salmonella sp was observed by measuring the turbidity level on the microplate at a wavelength of 630 nm.

Preparation of microbial cultivation and plant bioactive substances
Escherichia coli or Salmonella sp culture with a concentration of 108 CFU/mL bacteria was made through its turbidity setting at the absorbance of 0.138 using a spectrophotometer at 620 nm wavelength.The liquid smoke of cashew shells (CAM), meniran extract (EM), clove leaf extract (EDC), and bacitracin (Bac) before being used for an inhibitory test, were first filtered using a filter membrane with 0.45 μm pores.CAM is made with a concentration of 0.25 by mixing 1 mL of pure CAM with 3 mL of aqueous.EM is made with a concentration of 0.25 by mixing 1 mL of pure EM with 3 mL of aqueous).EDC was made with a concentration of 0.125 by mixing 0.5 mL of pure EDC with 3.5 mL of aqueous.The three extracts were then mixed with a ratio of 1 : 1 : 1 (100%), i.e 1 ml CAM + 1 ml EM + 1 ml EDC is called 100% extract combination (KE100).Then, this combination was made in three concentration levels with 50% dilution (KE50) and 25% (KE25), by adding aquadest with a ratio of 1 : 1 and 1 : 3.As a comparison, a positive control (K +) is used Zn-Bacitracin at a concentration of 250 ppm.Blank is made by mixing aqueous with nutrient broth medium (NBM) without E. coli or Salmonella sp bacteria in microplate reader wells.Negative control (K-) is made by adding bacteria in aqueous solution.For K +, K-and other treatments, a well-filled nutrient broth medium was mixed with 140 μL bacteria.
In summary, the bacteria inhibitory treatment performed is as follows: This study has fulfilled the code of ethics no.Balitbangtan/Balitnak/A/ 03/2016.For testing its effectiveness in vivo the concentration is increased twice for high doses and halved or to 50% for low doses.Testing the effectiveness of bioactive combination was also done using the powder form.The concentration of the ingredients in the form of flour uses the conversion 4 because 1 ml of the extract is obtained from 4 grams of flour.However, the CAM is not in the form of powder.Thus, the amount of each extract and powder added in the ration is presented in Table 1.

Chicken and management
Chicken used in this study was the broiler strain Ross maintained from day-old chick until 35 days.Feed and drinking water were given ad libitum.Chicken fed with the same composition, namely feed starter for 1-21 days old chickens and grower for chickens aged 22-35 days.The treatment consisted of 8 types of rations, each treatment consisted of 2 replications and each replication consisted of 5 DOCs.At the end of the experiment (35 days), blood was taken from 2 chickens on each replication.The basal ration composition was administered equally in all treatments, while the difference between treatments described with different levels of extract/powder combinations is described in Table 1.Body weight of the chicken weighed at the time of blood sampling (age 35 days).Then as much as 4 chickens from each treatment were euthanized to measure the weight of several internal organs (spleen and bursa fabricius).Intestinal contents were also taken for measuring the total amount of bacteria and the amount of E. coli.Calculation of total bacteria and E. coli was done 4 times in each treatment.Methods of calculating total bacteria and E. coli from the intestine were performed based on APHA procedure (2015).

Statistic analysis
Statistical analysis was performed by analysis of variance patterns Completely Randomized Design to compare between all treatments.Completely Randomized Design Factorial to compare between the extract and the powder form and between high, medium, and low doses.When variance analysis (ANOVA) is a significant difference between treatments at P<0.05 then continued with LSD test.

RESULTS AND DISCUSSION
The effect of combining CAM, EM, and EDC extracts on the growth of E. coli and Salmonella sp.
The effect of combining plant extracts on the growth of E. coli and Salmonella sp is shown in Figures 2 and  3. A 100% concentration of the combination of CAM, EM, and EDC more effective inhibits the growth of E. coli bacteria compared to 50% and 25% concentrations and treatment of Zn-bacitracin antibiotics.This result indicated that the combination of bioactive substances with high concentrations wasvery potent in inhibiting E. coli growth compared to concentrations of 50% and 25%.The concentration of a bioactive substance in medium (50%) and low (25%) was better inhibited the growth of E. coli and Salmonella sp than antibiotic zinc bacitracin (recommended dose for AGP).Therefore, for a combination of bioactive substances at a dose of 25% is sufficient to replace AGP.Several studies have shown that A. occidental plants contain active compounds of anacardic acid, cardanol, cardols, 2-methycardols 10-20% (Saidu et al. 2012;Setianto et al. 2009;Kumar et al. 2002).P. niruri L plants contain saponins, tannins, phenols, glycosides, flavonoids, and terpenoids (Gbadamosi et al. 2015), and S. aromaticum plants containing eugenol (Perez-Jiménez et al. 2010).Many types of active compounds present on these three types of plants (CAM, EM, and EDC) may have a role in the destruction of membranes, cell walls, and even cell nuclei of bacteria so that bacteria are unable to reproduce and eventually die (Harborne 1987; Turgis et al. 2009).
CAM has potential as a Gram-positive antibacterial (Himejima & Kubo 1991).The bioactive compound CNSL A. occidentale L. is able to penetrate the bacterial lipid bilayer membrane (Parasa et al. 2011), thus destroying the membrane bilayer.The same thing occurs to E. coli that cause E. coli defense to the outer environment to decrease, so E. coli can not develop.
Where in E. coli replication occurs every 20 minutes.The bioactive substance in CAM has the potential to inhibit the growth of E. coli so that the population declines.
The bioactive substances present in P. niruri L at various concentrations are able to inhibit E. coli growth by destroying the membrane structure (Monte et al. 2014;Gbadamosi et al. 2015).In vitro, at 60% concentration, no bacterial colonies of S. dysenteriae (Munfaati et al. 2015) were found.The concentration of 10 mg/ml inhibits E. coli growth with an inhibitory zone of up to 29 mm (Gbadamosi et al. 2015).The concentration of P. niruri extract 0.0313 g/ml could inhibit the growth of Edwardsiella tarda bacteria (Sudarno et al. 2011).At concentrations of 1000 mg/mL also inhibited E. coli, Staphylococcus aureus, and Salmonella typhi (Ekwenye & Njoku 2006).Water extract on P. niruri with a concentration of 0.75% can result in a 23 mm clear zone in E. coli (Lestariningsih et al. 2015).
Eugenol as the most dominant bioactive substance in S. aromaticum plant with water extract at 3% concentration able to inhibit the growth of E. coli, S. aureus and Bacillus cereus (Cortés-Rojas et al. 2014).S. aromaticum extract with ethanol fraction has 18 mm inhibition zone, while extract with methanol fraction has 20 mm clear zone to E. coli (Pandey & Singh 2011).S. aromaticum extract at a concentration of 2000; 1500; and 1000 ppm showed each clear zone on E. coli about 13 mm; 9 mm, and 7 mm, and at the same concentration showed clear zone on Salmonella typhi 23 mm; 15 mm; and 10 mm (Kumar et al. 2014).The results of this study indicated that the combination of CAM, EM, and EDC could inhibit the growth of E. coli and Salmonella sp.
Figure 1 showed that the bioactive substances CAM, EM, and EDC at the lowest concentration have responded to inhibit the growth of E. coli is stronger than Zn-bacitracin antibiotics dose 500 and 250 ppm.The ability of a combination of CAM, EM, and EDC to inhibit the growth of E. coli at a low concentration (25%) match the ability of antibiotics.Therefore, in its application, the use of low concentrations can reduce the cost of bioactive substances.
Combinations of CAM, EM, and EDC with concentrations of 100%, 50%, and 25% indicated inhibition of Salmonella sp growth were similar (Figure 2).Similarly, López et al. (2012) CAM on broilers inhibits Salmonella sp growth in the digestive tract.In the study of Sinurat et al. (2018) also reported that CAM may inhibit the growth of E. coli and Salmonella sp.Research on the use of liquid smoke from cashew shells against Salmonella sp has not been done extensively so that the information is still limited.
P. niruri L significantly decreased Salmonella sp population in spleens infected with Salmonella sp (Sunarmo 2009), At concentration 31.25-62.50mg/mL P. niruri L extract also decreased Salmonella sp (Ekwenye & Njoku 2006).Yusianti (2001) reported, in vitro use of P. niruri L extract at 0.6% concentration was able to inhibit the growth of Salmonella pullorum.Besides being a resistor of bacterial growth, P. niruri L extract was expected to increase the resistance of livestock body, because it has a high antioxidant ability (Sinurat et al. 2018).
The bioactive substances commonly found in plants are polyphenols, diterpene, alkaloids, and flavonoids (Rao et al. 2004).The delayed growth of E. coli and Salmonella sp is indicated because the bioactive substances of EM, CAM, and EDC are likely to form complexes with bacterial proteins through hydrogen bonds, consequently, the formation of nucleic acids and proteins in cells is inhibited.Turgis et al. (2009) stated that phenol (with-OH groups) can dissolve the lipids in the cell walls, thereby disrupting the cytoplasmic membrane performance and inhibit binding ATPase that cause cells to become lysis, as a result of bacterial growth is inhibited.Santoso (2008) reported, that the alkaloids contained in frangipani flowers (Plumeria acuminata) can suppress the growth of S. dysenteriae bacteria in vitro.From the results of in vitro assays, it is concluded that bioactive substances contained in the combination of EM, AM, and EDC are able to inhibit the total population of E. coli and Salmonella sp.

The effect of combination CAM, EM, and EDC on live weight, immune response, and blood profile
Body weight at the treatment of extract in medium and low dose, powder in high, moderate and low dose significantly (P<0.05) was significantly higher than negative control (Table 1).Combination of CAM, EM, and EDC in medium-dose at extract medium dose, powder in high, medium and low dose was not significantly different (P>0.05) with Zn-bacitracin treatment on live weight.But the treatment of medium and low dose extracts, high, medium and low-dose powder significantly (P<0.05)resulted in higher live weight than negative controls, which almost matched the antibiotic-fed chicken weight (Table 1).This result is only an early indication of the effect of the bioactive combination on chicken performance, since the number of tests and replication cattle used in this study is very limited.Tests using more numbers of livestock need to be done to determine the effect on livestock performance.
Giving combination CAM, EM, and EDC has the ability to replace antibiotics (Zn bacitracin), it was shown from the weight of life did not differ between them, while chicken negative control treatment showed lower body weight than other treatments.Attia et al. (2017) reported that a mixture of thyme extract 400 g, peppermint extract 300 g, green tea extracts 200 g, and licorice extract 100 g given to broiler chickens also showed improved performance compared to negative controls.The addition of a mixture of alfalfa leaf meal, cornflower, leaf senna and absinthe in broiler chicken feed also better than the control diet on enhances broiler growth (Khaligh et al. 2011).The mixture of some plants either in extract or flour form positively increases broiler weight, this also happens in combination CAM, EM, and EDC that can improve broiler performance.Bioactive substances of CAM such as phenol and anacardate act as antibacterials so that pathogenic bacteria in the intestine were eliminated and nutrients can be absorbed more optimally for growth needs.Khan (2010) reported that filantin, hypofilantin and flavonoids contained in EM act as a hepatoprotective agent (to prevent liver damage), thus metabolism in the liver such as protein synthesis for chicken growth is fulfilled.Giving eugenol as much as 100 mg/kg may improve the growth of chickens (Rychen et al. 2017).Likewise, eugenol contained in ED can increase chicken growth.Thus the combination of CAM, EM, ED has a positive synergistic effect on chicken performance.

Immune responses in chickens by administering a combination of plant bioactive
Statistical analysis of the relative weight of spleen and bursa fabricius and blood profile was not significant (P>0.05) between all treatments (Table 2).Spleen included in the secondary lymphoid functions to form granulocytes and erythrocytes.Relative spleen weights were not significantly different (P>0.05)among treatments, it was indicating the provision of a combination of CAM, EM, and ED at all doses did not affect the production of granulocytes, and erythrocytes in chickens.This indicates that when chickens are given a combination of CAM, EM, and EDC does not interfere with the production of granulocytes, and erythrocytes.Abdulkarimi (2011) reported that a single thyme administration did not affect the relative spleen weight.Giving mixed Echinacea purpurea, watercress, absinthe, and polygermander 10g / kg in broiler chicken feed had no effect on the spleen relative weight (Khaligh 2011).This indicates that the provision of plant material on the chicken does not affect the size of the spleen.
Bursa fabricius as the primary lymphoid organ with the humoral immune system plays a role in the synthesis and secretion of antibody substances into the blood circulation.In the combination of CAM, EM, and EDC the weight of the fabrication stock was not significantly different (P>0.05) with antibiotic treatment.The size of bursa fabricius affects the formation of antibodies, this indicates that the combination of CAM, EM, and EDC has a positive effect on antibody formation.Table 2 shows that although the relative weight of the bursa fabricius was not significant (P<0.05) between treatments, the combination of high and low powder produced a higher weight than antibiotic treatment.Likewise, Khaligh et al. (2011) reported that administration of a mixture of thyme extract 400 g, peppermint extract 300 g, green tea extract 200 g and licorice extract 100 g in broiler chickens showed the relative weight of the bursa fabricius there was no difference between both of them.Similarly, the administration of single thyme through drinking water does not show a difference with control (Abdulkarimi 2011).

Blood profile
The number of hematocrits, hemoglobin, lymphocytes, monocytes, and basophils were not significantly different (P>0.05)among all treatments (Table 2).This indicates that administration of a combination of CAM, EM, and EDC as a substitute for antibiotics do not interfere with the synthesis of hematocrit, hemoglobin, lymphocytes, monocytes, and basophils.Overall that antibiotic and bioactive combination treatments have no significant effect on the immune system.This can be seen from the size of the organ weight that does not affect the immune system.In fact, in vitro, the bioactive combination tested contained ingredients with a high antioxidant ability of P. niruri L, as reported by Sinurat et al. (2018).This is likely because the chickens that are raised are not challenged against diseases or bacteria.

Effect of the combination of CAM, EM, and EDC on total bacteria and number of Escherichia coli in the ileum
The combination of CAM, EM, and EDC in broiler chickens in the form of extracts or powder was not significantly different (P>0.05)against total bacteria and the number of E. coli in the ileum.However, with the medium dose of extract treatment showed the total bacteria was lower than Zn-bacitracin treatment, this indicates that the combination of CAM, EM, and ED in medium dose can replace antibiotics.Likewise, the number of E. coli is still lower than the control which indicates that the combination treatment of CAM, EM, and ED can replace Zn-Bacitracin (Table 3).The use of herb extracts as natural medicine has long been practised as an antimicrobial in the intestinal ecosystem to increase feed digestibility (Hernández et al. 2004).Attia et al. (2017) reported giving a mixture of oregano, fenugreek, chamomile and fennel extract reduced coliform bacteria and E. coli.The single administration of essential oil of C. xanthorrhiza or lemon peel did not affect the total bacteria in the ileum and cecum of broiler (Akbarian et al. 2013).

Effect of form and dose combination of CAM, EM, and EDC on total bacteria and number of E. coli
Treatment of combination of CAM, EM, EDC in broiler chickens between extract and powder form in high, medium, and low concentration was not significantly different (P>0.05) with Zn-Bacitracin treatment on total bacteria and the number of E. coli in the ileum (Table 4).This indicates that bioactive substances of three combinations (CAM, EM, EDC) have the same ability as Zn-Bacitracin as an antibacterial.Himejima & Kubo (1991) reported that CAM had the ability to kill Gram-positive bacteria, where the bioactive compound of CNSL Anacardium occidentale L. was able to penetrate the membrane of bacterial lipid bilayers (Parasa et al. 2011), thus damaging its layer.The same thing happened to E. coli as described earlier that the defense of E. coli against the external environment decreases due to damage to cell walls by bioactive substances so that E. coli in the ileum cannot develop.
While the bioactive substances contained in P. niruri L) such as alkaloids saponins, tannins, phenols, glycosides can inhibit the growth of E. coli by damaging the membrane structure (Monte et al. 2014;Gbadamosi et al. 2015).Reportedly, P. niruri L extract at various concentrations can inhibit bacterial growth.The use of P. niruri L extract in vitro at a concentration

CONCLUSION
The combination of cashew shell liquid of Anacardium officinale (CAM), Phyllanthus niruri L. (EM) extract, and clove leaf extract (EDC) can inhibit the growth of Escherichia coli and Salmonella sp.The best dose combination extract in inhibiting the growth of E. coli and Salmonella sp. is 100%, but the 25% dose has matched the AGP in vitro.In biological tests, a combination of CAM, EM, and EDC in the form of extracts with high, medium or low doses did not affect spleen weight, bursa fabricius, and blood profile.The best combination of CAM, EM, and EDC in the form of extracts or powder that reduces the total population of bacteria and the population of E. coli in the chicken intestine is a moderate dose.Likewise, for weight gain, there are indications that medium doses of extracts or powder can replace antibiotics.So it is concluded that the combination of CAM, EM, and EDC has the potential to replace AGP in poultry feed, especially on chicken.

Figure 1 .Figure 2 .
Figure 1.The effect of combining EM, CAM, and EDC extracts on E. coli growth.

Table 1 .
Combination dosage of the smoke of cashew shells (CAM), meniran extract (EM), And clove leaf extract (EDC) to be tested

the effectiveness of combinations of bioactive substances in feed
Combination of plant bioactive: The combination of plant extracts consists of a combination of the liquid smoke of Anacardium occidentale (CAM) shell, Phyllanthus niruri L. (EM) extract, and Syzygium aromaticum leaf extract (EDC).A combination with various concentrations was conducted to test its effectiveness when mixed in the ration.Determination of concentration in ration was based on effective concentration to suppress E. coli and Salmonella sp growth in previous research in vitro, that is combination with concentration 0.0625% CAM + 0.0625% EM + 0.0313% EDC.

Table 2 .
Effect of the combination of CAM, EM and EDC on life weight, the relative weight of spleen and bursa fabricius, and blood profile Description: Different subscripts in different lanes show significant differences (P <0.05)

Table 3 .
Effect of the combination of CAM, EM, EDC on total bacteria and number of E. coli in vivo