EFFECTIVENESS OF PROBIOTIC AND DIGESTIVE ENZYMES IN FERMENTED DIET ON GROWTH, FAT AND CHOLESTEROL CONTENTS OF BROILER

The present study aims to determine the effect of fermented diet containing probiotics (P) and digestive enzymes (DE) with different feed levels on growth, fat and cholesterol contents of broiler chickens by 5 treatments: P0= control (fermented diet without P and DE), P1= fermented diet contained 0.5% P only, P2= fermented diet contained 0.5% DE only, P3= fermented diet contained 0.5% P and 1% DE, and P4= fermented diet contained 1% P and 0.5% DE. A hundred DOCs of broiler chickens strain CP 707 were used (unsex) and it designed by a completely randomized design with 5 treatments, 4 replications and each replication consisted of 5 DOCs. The results of present study showed that broiler fed on fermented diet containing P and/or DE with different levels had a significant effect (P<0.05) on final body weight, weight gain, carcass weight, fat content on thigh muscle. Broiler fed on fermented diet contained 0.5% DE only (P2) resulted in better growth and weight gain than other treatments. However, fermented diet contained probiotics and digestive enzymes had no effect on fat and cholesterol contents in breast muscle. It was concluded that the addition of digestive enzymes in fermented diet formulated by local raw materials was very effective than probiotics in increasing body weight gain, carcass weight and prevented the increase in fat and cholesterol contents both in breast and thight muscles of broiler chicken.


INTRODUCTION
The use of fermented diet based on local ingredients in the broiler business is limited by low digestibility, high crude fiber and fat content that inhibit broiler growth. A strategy to increase the benefits of fermented diet on broiler chicken by using feed supplement and feed additives that have a positive effect on growth and meat quality. From previous research, it was known that the addition of probiotics in fermented diet has not provided maximum results to support optimal growth of broilers (Yaman et al. 2022). This condition is due to the local ingredients in fermented diet still containing high anti-nutrients (Bai et al. 2013;Alagawany et al. 2018;Andriani et al. 2020).
It was well known that probiotics has not been able to increase optimally protein digestibility of fermented diet formulated by local materials derived from agricultural waste due to it is bounded in conjugated protein form which is difficult to digest by chicken. Protein content of fermented diet was not metabolized maximally into limiting amino acids for broiler growth, especially methionine and lysine which are limiting amino acids for growth process of broiler chickens (Yaman et al. 2000;Kabir et al. 2004).
According to Zhai et al. (2014) and Arif et al. (2020), the anti-nutrient content in chicken feed can be suppressed by the addition of digestive enzymes in diet formulation. Digestive enzymes in poultry have the ability to increase the digestibility of rations by breaking down anti-nutritional compounds or complex compounds. Consequently, the availability of main nutrients of diet increase to stimulate chicken growth. In addition, digestive enzymes can help keep digestive tract of chicken healthy by helping digest lowdigestible protein which is bound in a complex form. Yegani and Korver (2013) reported that addition of digestive enzymes in chicken feed reduced antinutritional factors commonly found in raw materials of plant origin.
However, the effectiveness of the use of probiotics and digestive enzymes in non-conventional chicken diet (fermented diet) is dependent on type of raw materials, diet composition, nutrients content, ratio of probiotic : digestive enzyme in diet and health condition of digestive organs (Shirley and Edwards 2003;Amerah et al. 2013;Abd El-Moneim et al. 2019). More detailed information is still important reason and further research is required to ensure the effectiveness of probiotic and digestive enzyme in fermented diet on growth and meat quality of broiler chickens.

MATERIALS AND METHODS
The present study was carried out for 42 days at Laboratory of Animal Science, Faculty of Agriculture, Universitas Syiah Kuala, Banda Aceh. This study used 100-day old chick (DOC) of broiler strains CP 707, fermented diet, digestive enzymes and probiotics. The probiotic used contains lactic acid bacteria (Genus Lactobacillus, Entercoccus, Pediococcus, and Bifidobacterium), spore bacteria (Genus Bacillus) and yeast, while digested enzymes contain a combination of xylanse, amylase, protease and phytase enzymes as well as minerals. The addition of enzymes plays a role in increasing the digestibility of nutrients and reducing the negative effects of anti-nutritional compounds in feed.
All chicks placed in a postal cage equipped with a feeder, drinker, and brooder. Chicks were grouped on the different feeding treatments carried out using formulation of fermented diet, probiotics (P), and digestive enzymes (DE). Diet treatment consisted of commercial diet subtituted by fermented diet were P0= control (without P and DE), P1= containing 0.5% P and 0% DE, P2= containing 0% P and 0.5% DE, P3= containing 0.5% P and 1% DE, and P4= containing 1% P and 0.5% DE. The formulation and nutritional composition of fermented diet used in present study were listed in Table 1. The probiotic used in present study contained Lactobacillus acidophilus, Lactobacillus casei, Pediococcus sp, Bacillus cereus and Bacillus subtilis to stimulate the growth of broilers. Digestive enzymes used in fermented diet contain cellulose, glucanase, phytase, xylanase, amylase and protease. Feeding treatment was carried out in the morning at 08.00 WIB and in the afternoon at 17.00 WIB.
Drinking water was provided ad libitum. Body weight gain of chicken was calculated by reducing the body weight of the week concerned with the previous week. Final body weight was carried out by weighing the chickens at the end of rearing. Carcass weight was observed at the end of the rearing period. Feed conversion ratio was obtained by dividing the ration consumption by the weight gain. Cholesterol levels were measured with a spectrofluorophotometer RF-1501 using a commercial kit. The procedure for measuring cholesterol levels in breast and thigh muscles of chicken with a spectrofluorophotometer RF-1501 using a commercial kit after the supernatant was obtained from the enzymatic separation process using the Liebermen-Burchard method.

Final Body Weight, Weight Gain, Feed Consumption and Feed Conversion of Broilers
The results of present study showed that broiler fed on fermented diet containing probiotics and digestive enzymes with different feed levels had a significant effect on final body weight and weight gain. Broiler chickens consuming the fermented diet contained 0.5% of digestive enzyme resulted a higher body weight than control feeds and feeds containing probiotics or a mixture of probiotic and digestive enzyme ( Table 2).
In addition, fed on commercial diet substituted by fermented diet contained probiotic and digestive enzyme had a significant effect increased on final body weight and weight gain of broiler chicken. However, fermented diet containing probiotics and digestive enzymes had no significant effect on feed consumption and feed conversion. Probiotic containing Bacillus sp. produce lipase to reduce fat content in meat chicken (Patterson and Burkholder 2003). Feeding broiler on fermented diet containing probiotics and digestive enzymes did not have a negative impact on broilers in terms of weight gain. These results proved that the role of digestive enzymes is very effective in increasing the digestibility of local feed ingredients contained in fermented diet which can reduce factors such as antinutritional factors in feed ingredients (lectins and trypsin inhibitors) (Yegani et al., 2013). Compared to probiotics, digestive enzyme has a more important function in preventing the effects of antinutrients to bind protein and energy sources so that it can be easily digested by broilers. The effectiveness of adding digestive enzymes in this study was also seen from the increase in feed conversion value. In the P2 treatment, the feed conversion was better (2.09) than the control treatment (2.25) although not significantly different. This indicated that the addition of digestive enzymes is very effective in increasing the digestibility of feed ingredients that can be converted optimally into body weight. This is in line with the main function of digestive enzymes for poultry as a biological catalyst composed of proteins, amino acids with minerals and vitamins. It was clear that the advantages of using commercial enzymes in poultry feeds include improved productive performance and feed utilization, minimized environmental pollution due to reduced nutrient of manure (Cowieson et al. 2006). Phytases, carbohydrases, and proteases are most important enzymes that degrade crude fiber, starch, phytates, and protein which are eventually digested or excreted by the animal, having no residual effect on products like meat or egg. In addition, Slominski (2011) reported that carbohydrase enzymes include amylase and xylanase and are used to improve the digestibility of carbohydrates in feed ingredients of chicken diet composed by local materials.

Carcass Weight, Breast and Thigh Muscle Fat Content of Broiler
Carcass weight is closely related to fat accumulation in poultry especially on broiler. Patterson and Burkholder (2003) reported that fat accumulation was influenced by feed composition such as energy content in feed, ratio of protein energy and fat content of feed. Fat content of carcass is the result of the influence of nutrient intake which is stored in the cells as an energy reserve for chicken.
The results of this study indicated that the increase in fat content of thigh muscle of broiler fed on fermented diet with a mixture of probiotics and digestive enzymes was closely related to the decrease in poultry abdominal fat (Table 3). However, the fermented diet did not affect the fat content of the poultry breast muscle. This is because the breast muscle is very sensitive to changes in nutritional factors. In accordance with the opinion of Yaman et al. (2000) which states that the breast muscle is a fast twitch glycolytic muscle in chicken which is very sensitive to changes in the quality and quantity of feed. The fat content in the breast muscle is needed for membrane synthesis, carbohydrates, protein modification, the formation of structural elements in cells and tissues.
Overall, it was well known that fermented diet with the addition of digestive enzymes and probiotics has a positive effect on fat content because it produces a lowest fat level in the breast and thigh muscles compared to broiler chickens fed commercial feed (manufactured feed) which was between 2.97-5.12% (Loh et al., 2013). Fat percentage on both muscles were decreased by fed on fermented fed compared with the fat content of broiler chicken in each strain generally ranging from 15% to 20% (Ahmed et al. 2015). It was concluded that the addition of probiotics and digestive enzymes was not only able to increase body weight and carcass growth, but also was very good for the quality of chicken carcass without increasing the fat content of chicken muscle.

Cholesterol Content on Breast and Thigh Muscle
The results of the study on the addition of probiotics and digestive enzymes in fermented diet on cholesterol levels in breast and thigh muscles were observed at 6 weeks of age. The results of the measurement of breast and muscle cholesterol levels in broiler chickens due to fermented diet containing probiotics and digestive enzymes in different level in fermented diet did not show a significant effect on cholesterol level in both on breast and thigh muscles of broiler (Table 4). Cholesterol levels in the thigh and breast muscles of broiler chickens fed on fermented diet containing probiotics and digestive enzymes were in the normal range of 150-200 mg/dL.
In term of cholesterol content, the results of this study indicated that fermented diet containing probiotics and digestive enzymes did not increase cholesterol levels both in breast and thigh muscles. This result showed that the probiotic and digestive enzyme rich fermented diet was able to effectively use feed energy for the growth of broiler chicks. The amount of cholesterol in the meat is within a normal range, and there was no extra energy stored in the form of fat. Different superscripts in the same column indicated significant differences (P<0.05). P0= Control (feeding fermented diet containing 0% P and 0% DE), P1= Feeding fermented diet containing 0.5% P and 0% DE, P2= Feeding fermented diet containing 0% P and 0.5% DE, P3= Feeding fermented diet with containing 0.5% P and 1% DE, P4= Feeding fermented diet containing 1% P and 0.5% DE   Table 3. Carcass weight, breast muscle fat content and thigh muscle fat content of broiler chickens due to the addition of a combination of probiotics (P) and digestive enzymes (DE)  Different superscripts in the same column indicated significant differences (P<0.05). P0= Control (feeding fermented diet containing 0% P and 0% DE), P1= Feeding fermented diet containing 0.5% P and 0% DE, P2= Feeding fermented diet containing 0% P and 0.5% DE, P3= Feeding fermented diet with containing 0.5% P and 1% DE, P4= Feeding fermented diet containing 1% P and 0.5% DE It was known that cholesterol is an important molecule that plays a vital role in membrane structure, as well as being a precursor for the synthesis of molecules such as steroid hormones, vitamin D and bile acids (Loh et al. 2013). The concentration of cholesterol in broiler is depended on fat and fatty acid levels in muscle and adipose tissues. These components are the major factors that influence meat quality, particularly nutritional value and palatability (Coetzee and Hoffman 2002). Different in the dietary fatty acid (FA) composition would be reflected in the blood, which in turn could be transported to target organs such as muscle (Aghwan et al. 2014). In chicken, fat was deposited primarily under the skin, and chicken thighs are higher in fat and cholesterol than breast meat. For this reason, the function of probiotics and digestive enzymes indirectly becomes very important as part of poultry feed technology that will affect cholesterol content in broiler chicken meat. Fermented diet containing probiotic and digestive enzyme will greatly affect the quality of broiler chicken meat which is useful for supporting consumer health conditions.

CONCLUSION
Feeding a fermented diet with the addition of 0.5% digestive enzymes result in higher body and carcass weights in broilers. There was no change in fat content of breast muscle, but it was higher on thigh muscle compared to chickens fed commercial feed. However, the cholesterol levels of chickens fed on a fermented diet were still within the normal threshold, ranging from 150 to 200 mg/dL. It indicated that the addition of digestive enzymes was more effective than probiotics in increasing the benefits of a fermented diet in promoting the growth, carcass weight, and meat quality of broiler chickens. Table 4. Levels of breast muscle cholesterol and thigh muscle cholesterol levels of broiler chickens due to the activity of using a combination of probiotics (P) and digestive enzymes (DE) in fermented diet at 6 weeks of age