The usefulness of fermented katuk ( Sauropus androgynus ) plus bay leaves to modify fat accumulation , cholesterol and chemical composition of broiler meat

The usefulness of the addition of fermented herbs (katuk/Sauropus androgynusplus bay leaves) onfat accumulation, and chemical, amino acids and fatty acids composition ofbroiler meats was investigated.At 15 days of age, 280female broilers were divided into seven treatments, as follows:T0) the control;T1) broilers were given a ration with 2.5% fermented katuk plus bay leaf mixture formula 1;T2) broilerswere givena ration with 2.5% fermented katuk plus bay leaf mixture formula 2;T3) broilerswere givena ration with2.5% fermented katuk plus bay leaf mixture formula 3;T4) broilers were given a rationswith 5% fermented katuk plus bay leaf mixture formula 1;T5) broilers were givena ration with 5% fermented katuk plus bay leaf mixture formula 2;T6) broilers were givena ration with 5%  fermented katuk plus bay leaf mixture formula 3. The results of this study showed that the administration of fermented herbs to the ration significantly reduced abdominal fat, gizzard fat, total fat deposition, meat fat, and cholesterol contents. Their inclusions changed calcium, iron, moisture, serine, threonine, arginine, valine, lysine, methionine, myristic acid, pentadecanoic acid, palmitic acid, oleic acid, linolenic acid, cis-11,14-eicosadienoic acid, cis-5,8,11,14,17-eicosapentaenoic acid, and n-3 unsaturated fatty acids of female broiler meats. It can be concluded that administration of fermented herbs formula 2 at5% levels producedchicken meat with lowerfat and cholesterol levels, but higher protein, minerals and n-3 unsaturated fatty acidslevels.

The development of feed supplements from medicinal plant ingredients requires several medicinal plants to produce effective formulas.Kiyohara et al. (2004) state that formulas with many medicinal plants are expected to show a positive combination effect of many of these medicinal plants.Thus, the inclusion of katuk and bay leaf mixture may result in better meat quality than a single plant.However, katuk leaves and bay leaves contain high crude fiber and some antinutrient compounds and minerals that are not ready for use.For this reason, katuk leaves and bay leaves need to be improved in quality.One of a method to improve the quality of feedstuffs is fermentation.
Fermentation reduces anti-nutrition and crude fiber, improves nutrient values and feed utilization in poultry, ileal nutrients and amino acid digestibilities and mineral availability (Ahmed et al., 2014;Ari et al., 2012;Olaniyi and Mehhizadeh, 2013;Olagunyu and Ifesan, 2013;Sukaryana et al., 2011), and modifies lipid profiles (Santoso et al., 2010a).Santoso et al. (2015a) found that fermented katuk leaf reduced fat deposition in the liver and adipose tissue.Santoso et al. (2015b) reported that 5% cassava yeast fermented katuk leaf inclusion reduced fat and cholesterol contents, increased vitamin A, β carotene, protein and iron contents, and changes amino acid and fatty acid profiles in broiler meats.Lee at al. (2017) reported that the inclusion of fermented agricultural by-products reduced crude fat, but increased the protein content of broiler meat.
This research was done to analyze the use of fermented herbs (katuk-bay leaf mixture) on fat accumulation, cholesterol, proxymate composition, amino acid and fatty acid profiles in female broilers.

Fermentation of Katuk and Bay Leaves
The leaves obtained from the field or from traditional markets were air-dried for 5 days, and then dried under the sun for 1 hour for a dryness rate of about 10-12%, milled and stored in a plastic bag.The leaves powder was added water with 1:1 ratio, and then was cooked for 30 Meat Chemical Composition and Fermented Medicinal Herbs (U. Santoso et al.) minutes.The cooled leaves were then added with 0.5% cassava yeast and fermented for 24 hours at anaerobic condition.The fermented leaves were then dried under the sun, milled and stored in sealed plastic bag.

Animals and Diets
Seven hundred one day old chicks were given sugar contained drinking water to reduce stress due to travel.Brooder temperature was set according to standard maintenance procedure.At the age of 4 and 21 days, broiler chickens were vaccinated ND.At the age of 1-14 days, broiler chickens were fed commercial diets.At 14 days of age, 280 female broiler chickens were weighed and selected.
At 15 days of age, 280 female broilers were divided into seven treatments, as follows: T0) the control; T1) broilers were given a ration with 2.5% fermented katuk plus bay leaf mixture formula 1; T2) broilers were given a ration with 2.5% fermented katuk plus bay leaf mixture formula 2; T3) broilers were given a ration with 2.5% fermented katuk plus bay leaf mixture formula 3; T4) broilers were given a rations with 5% fermented katuk plus bay leaf mixture formula 1; T5) broilers were given a ration with 5% fermented katuk plus bay leaf mixture formula 2; T6) broilers were given a ration with 5% fermented katuk plus bay leaf mixture formula 3.

Sampling
At 34 days of age, six selected female broilers for each treatment were slaughtered.The accumulation of fat in the abdomen, gizzard, leg meats, heart, and neck were taken and weighed.To measure fatty liver scores, the colors of the broiler livers were compared with the standard colors from 1 to 5 (from dark brown (value 1) to yellowish white (value 5).The higher the value, the higher the fat content.The broiler thigh meats were taken, milled and frozen before analysis.

Laboratory Analysis
Fat content was analyzed by Soxhlet extraction; moisture content was analyzed by drying the samples at 105 o C; and protein content was analyzed using micro kjeldahl (Yerina (2015).Cholesterol levels were measured according to Liebermen-Burchrad with several modification as follows.0.1 gram of sample was added 10 mL of alcohol:hexane (3:1), heated and after cold centrifuged at 3.000 rpm for 10 minutes.The supernatant was separated, evaporated until a paste was formed.The paste was dissolved in chloroform and homogenized, and then added 2 mL of a mixture of sulfuric acid and acetic anhydrous (1:30)).After that it was placed in the dark room for 25 minutes until a green color was formed, read with a spectrophotometer with a wavelength of 420 nm (Marfuah, 2016).
Amino acid composition was measured by the method described by Ginting et al. (2017).Sixty milligrams of broiler thigh meat in erlenmeyer were added 4 ml of 6N HCl and refluxed for 24 hours at 110 ° C. The results of hydrolysis were neutralized with NaOH 6 N and filtered with 0.2 μm wattman paper.50μl was taken and 300 μL OPA (O-phthalaldehyde) solution and 1-2 drops of 2-mercaptoetanol was added, and stirred for 5 minutes.Furthermore, a sample of 20 μL was inserted into the HPLC injector alternately and ready for analysis.
Twenty gram broiler thigh meat was dried at 90 o C for 24 hours.The sample was extracted in soxhlet for 16 hours.The extraction was concentrated at 55 o C for 2 hours.A total of 0.0298 g of sample was added to 1mL of NaOH 0.5 N within methanol and heated over a water bath at 80 o C for 20 minutes.After cooling, 2mL BF3 was added and heated at 80 o C for 20 minutes, then cooled and added 2mL of saturated NaCl and 1 mL of hexane.The hexane layer was separated by a drop pipette and put in a tube containing 0.1 gram of Na2SO4 anhydrous, and left for 15 minutes.Sample solutions were separated and ready to be injected.The fatty acid composition was then measured by gas chromatography (Breuer et al., 2013).
Data obtained during the study were analyzed using one-way ANOVA.DMRT test was used if the measured variables had a significant effect at the 5% level (SPSS version 21).

Fat Accumulation
The Effect of fermented katuk and bay leaves on fat deposition in female broiler chickens is presented in Table 1.It was shown that the fat accumulation in the abdomen and gizzard was significantly affected (P<0.01),but the fat accumulation in sartorial, neck and heart was not affected.(P>0.05).The fatty liver score did also not changed.It was shown that T4 and T5 had lower abdominal fat than T1, T2, T3, and T6 but statistically similar to T0. T4 had lower gizzard fat than the other treatment groups.T4 had lower total fat deposition than T0, T1, T2, T3, and T6 but statistically similar to T5.The results showed that broilers fed a diet with fermented herbs 1 at the 5% level (T4) had the lowest total fat deposition.
The results showed that broilers fed a diet with fermented herbs 1 at the 5% level (T4) had the lowest total fat deposition.Lower fat deposition in T4 may be caused by active components presented in katuk and bay leaves such as flavonoids (Kamboh and Zhu, 2013), tannins (Selvi and Bhaskar, 2012), saponins (Afrose et al., 2010) and phenols (Qiong et al., 2014).Lower fat accumulation could be caused by several mechanisms such as lower pancreatic lipase activity (Mahmoud et al.,2013), and/or adipocyte size and number (Joo et al., 2010).Katuk and bay leaves are rich in flavonoids such as quercetin and kaempferol (Santoso et al., 2017).Flavonoids such as quercetin (Srobel et al., 2005) reduce adipocyte differentiation resulting in lower adipogenesis and thus resulting in lower adipocyte size and number.Phenolic compounds also reduce visceral adiposity by lowering adipocyte cell number and adipocyte cell size (de Melo et al., 2017).

Proxymate Composition of Meats
The Effect of fermented katuk and bay leaves on meat composition of female broiler chickens is presented in Table 2.This research showed that the addition of fermented herbs significantly affected fat, cholesterol, calcium, iron (P<0.05).phosphorus and moisture contents (P<0.01)but it did not significantly change protein and ash contents.Higher fat content was found in T0 as compared with T2, T3, T4, T5, and T6.T0 was found to consist of higher moisture content than T1, T2, T3, T4, and T5.T0 was found to have higher cholesterol content than T4, T5, and T6.T0 significantly had lower calcium content than T2 and T4.T0 significantly had lower phosphorus content than T2, T3, T4, T5, and T6.T0 significantly had lower iron content than T2.
The herb mixture was formulated by fermented katuk and bay leaves at the certain combination.Santoso et al. (2018) found that the addition of katuk leaf or bay leaf at 5% level reduced meat fat content by 26.7% or 10.0%, respectively.It was shown that the fermented katuk and bay leaves reduced meat fat content ranging from 22.5% to 45.2%.Broilers that provided a ration with 5% fermented herbs formula 2 had the lowest meat fat content.It appears that the combination of fermented herbs and feeding level influence meat fat content.A fermented product has been known to reduce the meat fat content in broiler chickens (Santoso et al., 2010a) .A reduction in the meat fat content by fermented product was partly caused by a reduction of fatty acid synthesis (Nie et al., 2015).Flavonoids (Zarrouki et al., 2010), tannins (Selvi and Bhaskar, 2012) and phenols (Qiong et al., 2014) have been reported to have anti lipid properties.Li and Tian (2004) also reported that flavonoids inhibited fatty acid synthase activity.
Low cholesterol in broilers provided a ration with 5% fermented herbs (T4, T5, T6) might be caused by saponins, flavonoids, alkaloids, and other compounds.Broilers fed 0.9% saponins decreased lipid digestibility and marked increases in cholesterol excretion (Jenkins and Atwal, 1994), whereas flavonoids decreased cholesterol and triglyceride contents of adipose and liver (Nan et al., 2014).Amani et al. (2014) found that an increase in flavonoid intake reduced the concentration of total plasma cholesterol.
This research showed that the addition of fermented herbs tended to increase protein content.Santoso et al. (2015) found that the addition of 2.5% or 5% fermented katuk leaf increased meat protein content.Santoso et al. (2018) also found that the addition of 5% katuk leaf powder increased meat protein content, whereas the inclusion of bay leaf tended to lower protein content.It has been established that fermentation improves protein values, protein digestibility and protein utilization, and ileal amino acid digestibilities in poultry (Ahmed et al., 2014;Cabuk et al., 2018;Sukaryana et al., 2011), and therefore increase protein retention in Meat Chemical Composition and Fermented Medicinal Herbs (U.Santoso et al.)  liver score.T0 = control; T1 = broiler chickens were fed diet with formula 1 at 2.5%; T2 = broiler chickens were fed diet with formula 2 at 2.5%; T3 = broiler chickens were fed diet with formula 3 at 2.5%; T4 = broiler chickens were fed diet with formula 1 at 5%; T5 = broilers were fed diet formula 2 at 5%; T6 = broilers were fed diet with formula 3 at 5%.  .026T0 = control; T1 = broiler chickens were fed diet with formula 1 at 2.5%; T2 = broiler chickens were fed diet with formula 2 at 2.5%; T3 = broiler chickens were fed diet with formula 3 at 2.5%; T4 = broiler chickens were fed diet with formula 1 at 5%; T5 = broilers were fed diet formula 2 at 5%; T6 = broilers were fed diet with formula 3 at 5%.An increase in phosphorus content of broiler meat in T2, T3, T4, T5, and T6, and improve calcium content in T2 and T4 might have a beneficial effect on human health.Calcium functions as muscle contraction and relaxation, transmission of nerve impulses, blood clots, regulating hormone secretion, co-factors in several enzymes, formation and maintenance of bone and teeth, smooth functioning of muscles, brain and nervous system (Shita and Sulistiyani, 2010), oocyte activation and fluid balance within cells (Praniva et al., 2013).Phosphate functions as skeletal mineralization (Takeda et al., 2004), intracellular signaling, membrane lipid component and the DNA backbone formation (Renkema et al., 2008).Furthermore, Plasma calcium and phosphate concentrations regulated the functions of various vital physiologic performances (Renkema et al., 2008).The tendency of higher meat iron content in broilers provided a ration with fermented herbs may be beneficial for a human.Iron has an important role in the transport of oxygen and electron, the synthesis of DNA, erythrocyte and hemoglobin, the conversion of blood sugar to energy, and the production of an enzyme (Gupta, 2014).Fermentation improves mineral bioavailability of mineral such as calcium, phosphorus and iron of feedstuffs (Gupta et al., 2015).Fermentation by yeast such as Saccharomyces cereviciae increases mineral bioavailability because of hydrolysis of phytate, folate biofortification and detoxification of mycotoxins (Hasan et al., 2014).Phytate forms strong chelate with calcium, magnesium, zinc, copper, iron and potassium.Thus, reducing phytate would result in better availability of phosphorus, calcium, magnesium, zinc, copper and potassium for broilers.

Amino Acid Profile
Table 3 shows the effect of fermented katuk and bay leaves on meat amino acid composition of female broiler chickens.The results of this research showed that fermented herb addition did not change aspartic acid, glutamic acid, histidine, glycine, alanine, tyrosine, phenylalanine, Ileucine, and leucine but significantly affected serine, threonine, arginine, valine, lysine (P<0.01),methionine and total amino acid (P<0.05).Serine, threonine and lysine contents of T1 were lower than the other treatment groups.Arginine levels of T1 and T6 were found to be lower than T0, T2, T3, T4, and T5.Methionine levels of T1, T4, and T6 were lower than T0, T2, T3, and T5.The highest valine level was found in T5.
The lower arginine, methionine, valine, serine, threonine and lysine in T1 are still not understood.It appears that feeding broilers with a diet containing fermented herbs formula 1 either at a level 2.5% or 5% did not benefit.Santoso et al. (2018) found that the addition of katuk or bay leaf powder increased or no change in methionine content.

Fatty Acid Profile
The effect of fermented katuk and bay leaves on meat fatty acid composition of female broiler chickens is presented in Table 4.These results showed that the addition of fermented herbs significantly influenced myristic acid, oleic acid, linolenic acid, cis-11,14-eicosadienoic acid, cis-5,8,11,14,17-eicosapentaenoic acid, saturated fatty acid, unsaturated fatty acid, n-3 unsaturated fatty acid (P<0.01),n-6 unsaturated fatty acid (P<0.01),total fatty acids (P<0.01),pentadecanoic acid and palmitic acid (P<0.05).Myristic acid of T1 was lower than T0, T4, T5, whereas pentadecanoic acid of T1 was lower than T0 and T5.Palmitic acid level of T1 was found to be lower than T0, T2, T3, T4, and T6.Oleic acid level of T1 was found to be lower than T0, T2, T3, T4, T5, and T6, whereas that of T5 was found to be lower than T0.Linolenic acid levels of T1 and T3 was found to be lower than T4 and T5.Cis-11,14-eicosadienoic acid level of P0 was found to be the lowest, whereas that of P5 was the highest.8,11,14,17-eicosapentaenoic acid level of T4 was found to be lower than T0, T1, T2, T3, and T5.Saturated fatty acid, unsaturated fatty acid, n-9 unsaturated fatty acid, and total fatty acid levels of T1 was the lowest.
Lower total fatty acid levels in T1 as compared with T0 is still not understood.Li and Tian (2004) reported that flavonoids inhibited fatty acid synthetase activity, a rate limiting enzyme for fatty acid synthesis in poultry, and thus resulting in lower fatty acid levels.Lower fatty acid levels might cause lower myristic acid, palmitic acid and oleic acid.Santoso et al. (2018) reported that feeding katuk leaf or bay leaf at 5% had no effect on myristic acid, pentadecanoic acid, palmitic acid, linolenic acid, cis-5,8,11,14,17-eicosapentaenoic acid, and oleic acid.Fermentation and the combination of those herbs may change that pattern, because the present study showed that .048T0 = control; T1 = broiler chickens were fed diet with formula 1 at 2.5%; T2 = broiler chickens were fed diet with formula 2 at 2.5%; T3 = broiler chickens were fed diet with formula 3 at 2.5%; T4 = broiler chickens were fed diet with formula 1 at 5%; T5 = broilers were fed diet formula 2 at 5%; T6 = broilers were fed diet with formula 3 at 5%.
fermented katuk-bay leaves inclusion affected the above fatty acids.Santoso et al. (2015b) found that myristic acid was increased in broiler chicken fed a diet with fermented katuk leaf at 2.5%, but its content was reduced when broiler chickens fed a diet with fermented katuk leaf powder at 5%.This pattern was in contrast with the present study, which showed lower myristic acid content when broilers were fed a diet with fermented herbs at 2.5%, but showed higher myristic acid content when broilers were fed a diet with fermented herbs at 5%.Thus, fermented herbs inclusion changed the respond of broiler chickens.Santoso et al. (2015b) reported that the inclusion of fermented katuk leaf powder reduced oleic acid and linoleic acid, whereas in the present study showed that the inclusion of fermented herbs reduced oleic acid but not linoleic acid.Thus, the addition of fermented herbs changed meat fatty acid profiles.Kamboh and Zhu (2013) reported that an increase in levels of flavonoid intake improved the ratio of n-6 to n-3 fatty acids and polyunsaturated fatty acids to saturated fatty acids in breast meats.

CONCLUSION
The inclusion of fermented katuk plus bay leaves reduced fat and cholesterol contents but increased or tended to increase protein, calcium, phosphorus, and iron contents and changed amino acid and fatty acid compositions in female broiler chickens.

Table 1 .
The Effect of Fermented Katuk and Bay Leaves on Fat Deposition in Female Broiler Chickens

Table 2 .
The Effect of Fermented Katuk and Bay Leaves on Meat Composition of Female Broiler Chickens Meat Chemical Composition and Fermented Medicinal Herbs (U.Santoso et al.)89

Table 3 .
The Effect of Fermented Katuk and Bay Leaves on Meat Amino Acid Composition of Female Broiler Chickens

Table 4 .
The Effect of Fermented Katuk and Bay Leaves on Meat Fatty Acid Composition of Female Broiler Chickens

Table 4 .
The Effect of Fermented Katuk and Bay Leaves on Meat Fatty Acid Composition of Broiler chickens (continued) ab 0