Efficacy of Moringa oleifera Lam. extracts and Pediococcus pentosaceus, Lactobacillus acidophilus, Lactobacillus plantarum probiotic during starter period on growth performance of male broiler chicken [version 3; peer review: 1 approved, 1 approved with reservations]

Background : Probiotics and medicinal plants have been used to support human and livestock health. This research aimed to evaluate the efficacy of Moringa oleifera Lam. leaf extract on the growth of P. pentosaceus, L.acidophilus and L. plantarum during starter period on nutrient intake, body weight gain, FCR and feed efficiency in broiler chicken. Methods: This study consisted


Introduction
Feed comprises ingredients provided to poultries to meet necessary dietary requirements for livestock growth, development, and reproduction. Breeders administer antibiotic growth promoters (AGP) to improve production, trigger growth, and act as an antibacterial. 1 However, the use of these growth promoters in animal production is gradually being restricted and banned in some countries. One antibiotic substitution which can be used in feed is probiotics.
Phytobiotics are feed additives derived from pure plant materials. Phytobiotics are able to control micro-organisms in the digestive tract of poultry. Phytobiotics are able to increase metabolic activities in the body, so these phytobiotics can be used as feed additives in poultry. Phytobiotics have proven to have several functions, such as a growth-promoting effect, antimicrobial activity, anti-inflammation activity and improving performance. [2][3][4][5] They allows to control micro-organism growth in the digestive tract of poultry by increasing the metabolic activities in the body, making them potential feed additives in poultry. 6,7 Pediococcus pentosaceus is a lactic acid bacteria (LAB). LABs are the bacterial group that ferments carbohydrates into lactic acid. 8 Probiotics are microorganisms capable of improving growth and feed efficiency. Several probiotics improving production performance in broilers are P. pentosaceus, Bifidobacterium sp, Lactobacillus casei, and Lactobacillus acidophilus. 9-14 P. pentosaceus is a Gram-positive bacteria, round-shaped, non-motile, not generating spores, and has negative catalase. As it grows, P. pentosaceus generates lactic acid and pediocin. 15 Pediocin is a bacteriocin generated by P. pentosaceus, which, in a sufficient amount, can eliminate pathogenic bacteria such as Listeria monocytogens, Staphylococcus aureus, Escherichia coli, Vibrio alginolyticus, Pseudomonas stutzeri, and Aeromonas. P. pentosaceus bacterial growth requires nutrition with carbon, nitrogen, and mineral sources. [16][17][18] Moringa oleifera belongs to the family Moringaecea. M. oleifera is very useful as a feed supplement for animals, as its leaves are highly nutritious. Moringa leaf carbohydrates reach 38.2 g for 100 g of Moringa leaf powder. 19 Moringa also contains proteins, fats, fibers, calcium, magnesium, phosphorus, potassium, copper, iron, vitamin B1, vitamin B2, vitamin B3, vitamin C, vitamin E, and essential amino acids, e.g., arginine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, and cysteine. 20 Probiotics containing L. acidophilus, L. casei, L. lactis, and Bifidobacterium spp. and M. oleifera extract can increase the production performance of Peking ducks. 21 Moringa leaves are antioxidant, antibacterial, anti-inflamatory, 22 and are rich in fats, proteins, vitamins, and minerals. Moringa leaves contain diversified phytochemicals such as flavonoids, saponins, tannins, phenols and alkaloids. 22, 23 The high-nutrient profile of Moringa suggests potential growth promoter and immunomodulatory effects. 24 Moringa can be used as a source of micronutrients and as a dietary supplement in poultry. 25 Nutrition consumed by poultry is used for maintenance and production. Feed efficiency in broilers is influenced by the level of feed consumption and body weight gain. High feed efficiency is produced due to low feed intake followed by a high rate of body weight gain. A high level of feed efficiency can reduce livestock production costs. 6,7,26,27 The study aimed to analyze the phytochemical properties of Moringa leaf extract and the effectiveness of M. oleifera extract with different dosages and incubation times on in vitro bacterial growth of P. pentosaceus, L. acidophilus and L. plantarum probiotic, as well as their effect on growth performance on broiler chicken starter phase in vivo.

Methods
This study was conducted in the Faculty of Veterinary Medicine, Universitas Airlangga, Surabaya. The trial design was a complete randomized design consisting of four treatments: P0, P1, P2, and P3, with five replications.
Moringa was extracted by mixing macerated moringa leaf with ethanol 70% and 96% as the solvent. Extraction with ethanol 70% can extract oligosaccharides. 28 The phytochemical screening test was carried out to determine the presence of the flavonoid, tannin, saponin, triterpenoid and alkaloid.

REVISED Amendments from Version 2
We have revised the abstract as suggested by the reviewers.
The phytochemical screening test of M. Oleifera extract showed the presence of flavonoids, saponins, tannins, triterpenoids and alkaloids.
Any further responses from the reviewers can be found at the end of the article Bacterial cell count measurement was conducted to measure the bacterial cell count of P. pentosaceus, L. acidophilus and L. plantarum before adding Moringa leaf extracts. A bacterial suspension comparison was performed using the McFarland 0.5 scale standard. After obtaining the same turbidity, a gradual dilution was conducted up to seven times to obtain a 10 1 bacterial concentration.
A sterile test tube was filled with Aquadest 9 mL mixed with Moringa leaf extracts with the following treatments: P0: Without M.oleifera extracts; P1: 0.1% M.oleifera extracts; P2: 0.2% M. oleifera extracts; P3: 0.3% M. oleifera extracts. Furthermore, each test tube was added with 1 mL of each isolate and incubated for 24 h at 37°C, after each isolate bacteria was grown in the Agar MRS media using the pour plate method and incubated for 12 and 17 h. The growth of probiotic bacteria was calculated for the colonies.

Statistical analysis
All data collected during this study were statistically analyzed under a completely randomized design. All data were tested for distribution normality and homogeneity statistics (analysis of variance). The data were analyzed using Statistical Design for Social Sciences (SPSS) v.22 (IBM Corp., NY, USA). Differences among means were detected using a one-way analysis of variance. The differences among means were determined using Duncan's test (p < 0.05).

Results
Phytochemical screening of M. oleifera extract Moringa leaf extract was tested for its chemical composition using a phytochemical screening test. At this stage, five kinds of tests were carried out: flavonoid test, tannin test, saponin test, triterpenoids test, and alkaloid test. The results of the phytochemical screening tests are presented in Table 1. The results of positive flavonoid test indicated by change in color, there was a change in color to orange. The interpretation of positive tannin, indicated by the color of the filtrate changed to a dark green-black color and the presence of saponin indicated by the foam. The interpretation of flavonoid, tannin , saponin were indicate in Figure 1A, B, C. The Figure 2 showed the result of positive triterpenoid test and the Figure 3 showed the result of alkaloid Test [Mayer reagent (-), Bouchardat reagent (+) and Wagner reagent (+).

Evaluation of several doses of M. oleifera extract
The growth results of the probiotics P. pentosaceus, L. acidophilus and L. plantarum with several levels of M. oleifera extract showed significant differences between the treatments (p < 0.05) (Table 2, Figure 4). Evaluation of probiotics, M. oleifera and combination of probiotics + M. oleifera in vivo The result of feed intake and nutrient intake of dry matter, ash, crude protein, crude fat, crude fiber and organic matter listed in Table 3. There was no significant difference (p > 0.05) between the treatments of feed intake, dry matter intake, ash intake, crude protein intake, crude fat intake, crude fiber intake, and organic matter intake in this study.     The result of body weight, daily body weight gain, feed conversion ratio, and feed efficiency are listed in Table 4. Body weight differed significantly (p < 0.05) between treatments. The control (T0) had the lowest body weight, which was different from all treatments. The average daily weight gain differed significantly (p < 0.05) between treatments. The control (T0), which differed from all treatments, had the lowest body weight gain. The FCR results revealed a significant difference (p < 0.05) between treatments. The feed efficiency results revealed a significant difference (p < 0.05) between treatments. T0 had the lowest feed efficiency value found.

Discussion
Phytochemical screening of M. oleifera extract The phytochemical screening revealed flavonoids, tannins, saponins, terpenoids, and alkaloids which could serve as a natural source of antimicrobials. The results obtained from the flavonoid test were a change in color. There was a change in color to orange ( Figure 1A). The orange to red color change is caused by the formation of flavilium salts. This indicates that the sample contained flavonoids.
In the tannin test, a positive result is indicated by a change in the color of the filtrate to green or blackish blue. In this test, the color of the filtrate changed to a dark green-black color ( Figure 1B), so the sample was considered test-positive for tannin. The test for the presence of saponin was indicated by the appearance of foam. The foam indicated the presence of glycosides which have the ability to form froth in water as they hydrolyze into glucose and other compounds. In the saponin test carried out, foam formed for approximately 10 minutes; with the addition of one drop of hydrochloric acid, the foam did not disappear and the sample tested positive for saponin ( Figure 1C).
The addition of concentrated sulfuric acid causes water to hydrolyze, which reacts with acetyl derivatives to form a red ring. A positive test for the presence of triterpenoids is indicated by a color change to red or purplish red. In the test conducted, the color changed to red (Figure 2), therefore the sample tested positive for triterpenoid.
The alkaloid test in this study used three reagents: Mayer's reagent, Bouchardat's reagent and Wagner's reagent. The test with Mayer's reagent showed no white precipitate ( Figure 3A), so the result is negative. With the Bouchardat reagent, a precipitate appeared and the color changed to orange ( Figure 3B), therefore the result is positive. With Wagner's reagent, a brown precipitate appeared ( Figure 3C). The precipitate was indicated as a potassium-alkaloid. Of the three reagents used, two of them were positive, so it can be concluded that Moringa leaves contains alkaloids.
These results are in accordance with the research of Kandeepan et al. which showed that the results of phytochemical analysis of M.oleifera revealed the presence of alkaloids, flavonoids, saponins, tannins and terpenoids. M. oleifera leaf extracts contain a substantial amount of phenolic chemicals, which are principally responsible for antioxidant activities. Secondary metabolites with bioactive properties. M. oleifera leaves continue to be excellent sources of micronutrients and phytochemicals for the creation of nutraceuticals and functional foods. 32 Secondary metabolites extracted from M.oleifera leaves using chloroform, ethyl acetate, and ethanol contain bioactive substances such as such as steroids, saponins, tannins, flavonoids, terpernoids and phlobatannins. 33 Evaluation of several doses of M. oleifera extract The lowest growth of P. pentosaceus, L. acidophilus and L. plantarum was found for the treatment without M. oleifera extract (P0), which was different from all treatments. The fastest growth of P. pentosaceus was found with the addition of 0.2% M. oleifera extract (P2), followed by 0.3% M. oleifera extract (P3) and 0.1% M.oleifera extract (P1). The highest growth of L. acidophilus was shown with the addition of 0.3% M. oleifera extract (P3), followed by the addition of 0.2% M. oleifera extract (P2) and 0.1% M. oleifera extract (P1). The highest growth of L. plantarum was shown in the 0.3% M. oleifera extract (P3) treatment, which was not different from 0.2% M.oleifera extract (P2), while P2 was not different from 0.1% M. oleifera extract (P1) ( Table 2).
Bacterial growth of P. pentosaceus L. acidophilus and L. plantarum was significantly improved after the addition of 0.1%, 0.2%, and 0.3% M. oleifera extract. This shows that the higher the M. oleifera extract concentration, the faster the bacterial growth of P. pentosaceus, L. acidophilus and L. plantarum. Microbial count difference was determined by the fermentation duration and nutrition availability. Primary nutrition required by P. pentosaceus, L. acidophilus and L. plantarum bacteria were carbon and nitrogen sources. Bacteria use carbon sources as the energy source and produced lactic acid while nitrogen is used to generate bacterial cell biomass. 34 M. oleifera extract contains the monosaccharides mannose, arabinose, xylose, and the oligosaccharides raffinose and stachyose. 35 These oligosaccharides can be used as nutrition for bacterial growth. 36 P. pentosaceus bacteria is an amylolytic bacterium that generates amylase and can hydrolyze carbohydrates into glucose. Glucose is a carbon source for bacteria as the energy fueling their growth and lactic acid formation. P. pentosaceus bacteria use oligosaccharides and monosaccharides in Moringa leaf as the energy source for fermentation. 37 P. pentosaceus is also classified into the proteolytic lactic acid bacterial group. Proteolytic bacteria are those capable of generating the protease enzyme to hydrolyze polypeptides in media into amino acids. Nitrogen is a constituting component of amino acids. Thus, bacteria can use amino acids as a nitrogen source for bacterial growth and DNA/RNA synthesis. Bacterial growth of P. pentosaceus on Agar MRS media ferments carbohydrates from Moringa leaf extracts into glucose and generate lactic acid. Metabolites of lactic acid are generated from glucose fermentation through glycolysis. P. pentosaceus metabolizes glucose into produce pyruvic acid through the Embden-Meyerhof pathway, which is then reduced into lactic acid by the lactase dehydrogenase enzyme and nicotinamide adenine dinucleotide phosphate (NADP).
High bacterial growth followed the high lactic acid concentration on the fermentation media. The pH reduction and total acid level increase on the fermentation media is beneficial since it hinders pathogenic microbes. The growth of P. pentosaceus bacteria produces pediocin, a bacteriocin that inhibits the growth of pathogenic bacteria. 10 P. pentosaceus incubated with M. oleifera extract could be used to improve the nutritional quality of rice bran. 38 M. oleifera extract's beneficial effects as phytobiotics are considered to be due to their antioxidant properties. Phytochemical compounds such as flavonoids, saponins and tannins, exhibit antimicrobial activity. 39 The phytochemical screening results in this study revealed flavonoids, tannins, saponins, triterpenoids, and alkaloids.  Table 4).
The results of this study are in accordance with other studies that showed the administration of Lactobacillus probiotic can improve gut health and contributes to increased growth performance. 40 Probiotics play a role in modulating the balance of microbiota in the digestive tract and in developing intestinal health so that they affect feed consumption, digestibility, absorption of nutrients and improve feed conversion ratio and feed efficiency. The use of probiotics in poultry also plays a role in modulating the immune response, maintaining the health of the intestinal tract and influencing stress reduction. [41][42][43] Other studies have shown that the use of L. acidophilus poses an obstacle to the invasion of pathogenic bacteria and modulates the immune response in vitro and in vivo. This is because L. acidophilus produces bacteriocin. Additionally, probiotics that are classified as lactic acid will produce acid thereby lowering the pH in the intestine. In poultry, the use of L. acidophilus can balance the intestinal microflora and reduce the proliferation of pathogenic bacteria through antagonism and competitive exclusion. 40,44 Protein availability can be increased by taking probiotics. Probiotics can improve the host's health by increasing intestinal villi size and nutrient uptake. 45 The use of probiotics can modify the intestinal ecosystem by decreasing pH through acid production by lactic acid probiotic bacteria and modulating enzyme activation in the digestive tract. 46,47 Several factors can affect the colonization of probiotic microbes in the intestine depending on the availability of fermented substrates (prebiotics), pH in the intestine, frequency and dosage of probiotics, genetics, age, health, stress factors and the nutritional status of the host. 48,49 This relates to the ability of probiotic micro-organisms to secrete enzymes such as protease, lipase, and amylase to help digest proteins, fats, and starch so that they can increase the availability of nutrients and help increase the digestion of feed nutrients. 50 M. oleifera extract contains phenolic compounds in insoluble and soluble bound forms. The bound phenolic compounds cannot be absorbed by the small intestine because they are bound by insoluble macromolecules such as cellulose, hemicellulose, structural proteins and pectin; these compounds enter the large intestine (colon), a fermentation by the intestinal microbiota occurs, which releases bound phenolic compounds. The released phenolic compounds can decrease pH, inhibit the growth of pathogenic bacteria and modulate the development of fermentative microflora so that it can improve host health. 51,52 Moringa has no toxic effects and does not interfere with nutrient absorption. 53 In this study, giving probiotics, M. oleifera extract and a combination of probiotics and M. oleifera extract during the starter phase of broiler chicken increased body weight and average daily weight gain, increased feed efficiency and improved feed conversion compared with control. This is because the addition of probiotics to poultry can modulate the balance of microbiota in the intestine, increasing feed digestibility and nutrient absorption. This is also influenced by the viability requirements of probiotics, where probiotics must be able to survive and not be damaged during processing to storage. The addition of probiotics increases nutrient bioavailability and improves poultry feed conversion. Probiotics must be able to survive various stress factors during processing, storage and digestion. Several different probiotic strains can exhibit wide variations in their abilities, such as efficacy as probiotics, stability of process and functional properties at the targeted site. 54 The phytoconstituents present in M. oleifera are flavonoids, niazirinin, niazirin, proanthocyanidin, anthocyanins, kaempferol-3-O-(6"-malonyl-glucoside), β-sitosterol, β-sitosterone, 4-hydroxymellein, octacosanic acid. 55 M. oleifera with ethanol extraction has a strong antioxidant activity. 56 Hydro-alcoholic extract of Moringa leaves contain 21.1% polysaccharides (% of dry matter). 53 The addition of a combination of probiotics and M. oleifera extract also resulted in better body weight gain, FCR and feed efficiency than the control. This is due to their proanthocyanidins content, which are condensed tannins (epicatechins and catechins) found in plants that provide protection against biotic and abiotic stresses, and act as antimicrobial, antioxidant, anti-inflammatory, vascular and cardiac activity. 57-59 The use of M. oleifera extract improved FCR and increase feed efficiency due to the presence of secondary metabolites produced by the plant, which also play a role in giving flavor to feed, and are influenced by their ability to form complexes with macromolecules (polysaccharides and proteins) and metal ions. 59,60 The use of probiotics and M. oleifera extract for feed efficiency agrees with Nikpiran et al. (2013) 61 who found a better FCR with the addition of prebiotics to broiler rations compared to controls. This is thought to be related to the availability of substrates and the presence of a better balance of microbiota in the gut, resulting in better feed efficiency. 62,63 The finding of increased average daily gain and improvement of FCR in this study by administering M. oleifera leaf extract to broiler chickens agrees with a previous study where the FCR value obtained was 1.41-1.65 and the average daily gain 62.52-70.60 g, while research by Akhouri (2013) using M. oleifera extract in starter phase broilers produced an FCR of 1.77 and average daily gain of 52.57 g. 64 The results of our study are also in line with Sjofjan's (2021) that the level of probiotics increased body weight gain, body weight, feed intake of broilers and improved FCR. 65

Conclusions
The phytochemical tests showed flavonoids, tannins, saponins, tripenoids, alkaloids. The use of M. oleifera extract at doses of 0.1%, 0.2%, and 0.3% increased the growth of P. pentosaceus, L. acidophilus and L. plantarum bacteria in vitro. The use of probiotics, M. oleifera extract and a combination of probiotics + M. oleifera extract used in vivo can improve the growth performance of starter-phase broilers chicken. Based on these results, the use of probiotics, M. oleifera extract or combination of both could be used as alternative antibiotic growth promoters in the poultry industry.   I recommend that the Article should be Approved for indexing.

If applicable, is the statistical analysis and its interpretation appropriate? Partly
Are all the source data underlying the results available to ensure full reproducibility? Partly Are the conclusions drawn adequately supported by the results? Partly broilers is influenced by the level of feed consumption and body weight gain. High feed efficiency is produced due to low feed intake followed by a high rate of body weight gain. A high level of feed efficiency can reduce livestock production costs 6

Revised:
The study aimed to analyze the phytochemical properties of Moringa leaf extract and the effectiveness of M. oleifera extract with different dosages and incubation times on in vitro bacterial growth of P. pentosaceus, L. acidophilus and L. plantarum probiotic, as well as their effect on growth performance on broiler chicken starter phase in vivo. Method: Correct to; "Phytochemical screening test was carried out to determine the presence of Flavonoid, tannin..." ○ Revised: The phytochemical screening test was carried out to determine the presence of the flavonoid, tannin, saponin, triterpenoid and alkaloid. Result: Good; but you need to put all the results together; some part of your result interpretation was added to the discussion.

Revised:
The results of positive flavonoid test indicated by change in color, there was a change in color to orange. The interpretation of positive tannin, indicated by the color of the filtrate changed to a dark green-black color and the presence of saponin indicated by the foam. The interpretation of flavonoid, tannin , saponin were indicate in Figure 1A, B, C. The Figure  2 showed the result of positive triterpenoid test and the figure 3 showed the result of alkaloid Test [Mayer reagent (-), Bouchardat reagent (+) and Wagner reagent (+). Discussion: Result interpretation (Fig 1A, 2, 3) should be part of results. Cut the result interpretations and add to the Results part. Try to comparing your findings with the findings of previous researchers. Secondary metabolites with bioactive properties. M. oleifera leaves continue to be excellent sources of micronutrients and phytochemicals for the creation of nutraceuticals and functional foods 32 . Secondary metabolites extracted from M. oleifera leaves using chloroform, ethyl acetate, and ethanol contain bioactive substances such as such as