Abstract: Objective: The present study was conducted to evaluate the effect of fermented Sauropus androgynus leaf extract on the chemical composition of broiler meat. Methodology: One hundred broilers aged 15 days were distributed into five treatment groups. Each treatment group was represented by four replicates of five broiler chickens each. The five treatment groups were as follows: (1) Broiler chickens were fed diet without fermented Sauropus androgynus leaf extract (FSALE) as the control (P0), (2) Broiler chickens were fed a diet supplemented with 4.5 g FSALE kg–1 diet (P1), (3) Broiler chickens were fed a diet supplemented with 9 g FSALE kg–1 diet (P2), (4) Broiler chickens were fed a diet supplemented with 13.5 g FSALE kg–1 diet (P3) and (5) Broiler chickens were fed a diet supplemented with 18 g FSALE kg–1 diet (P4). Results: The extract contained 25.46% protein, 1.34% fat, 3,642.6 μg β-carotene g–1, 8.17 mg iron g–1 and 0.17 mg sterol/100 mg. The extract was rich in palmitic acid (29.96%) and glutamic acid (2.221%). The results showed that FSALE inclusion significantly increased the contents of protein (p<0.01), β-carotene (p<0.001) and iron (p<0.001) of broiler meats, but it significantly reduced the contents of fat (p<0.05) and cholesterol (p<0.05) of broiler meats. The FSALE inclusion significantly reduced oleic acid (p<0.05) and increased docosahexaenoic acid (p<0.001). In addition, FSALE significantly reduced aspartic acid, serine, glycine, histidine, arginine, threonine and cystine (p<0.01), but FSALE significantly increased glutamic acid, alanine, proline, tyrosine, valine, methionine, phenylalanine and lysine (p<0.001). Conclusion: The FSALE inclusion at level of 4.5-18 g kg–1 in the diet increased the contents of iron, protein and β-carotene but it reduced the content of cholesterol in broiler meat. In addition, FSALE inclusion changed the composition of amino acids and fatty acids.
INTRODUCTION
The presence of a positive correlation between the concentration of fats such as cholesterol in the blood and the risk of atherosclerosis, coronary heart disease, stroke and other metabolic diseases1,2 encourages the broiler industry to produce low-fat meat. In addition, the reduction of fat deposition in depots is very important for the industry, because fat depots have lower prices than carcasses.
The total depot fat in broiler carcasses is 6%, which will decrease the industry profits as much as 4-6%. Sauropus androgynus leaves and their extracts have been shown to decrease fat deposition in broiler and layer chickens3,4. However, a decrease in fat deposition by Sauropus androgynus leaves and the extract is still low, thus it may be not effective when it is applied on a commercial scale5.
The tendency of weight loss in broiler chickens by Sauropus androgynus leaves6 will reduce the economic profit of the livestock industry. Sauropus androgynus leaves have some antinutrients such as oxalate, saponins, tannin and oligosaccharides7 and they can cause lung disorders8. Therefore, a method to improve nutrient contents and to reduce the antinutrient substances of the leaves is required. It has been established that fermentation improves feed nutrients and breaks down protein into simpler compounds such as peptides and amino acids, lowering crude fiber9-12 and reducing the contents of antinutrients such as tannins, oligosaccharides, phytic acid, phenol, saponins, oxalate, phytin phosphorus11,13-16, phenolic compounds and alkaloids14.
The results of previous studies17-19 have shown that the supplementation of Saccharomyces cerevisiae fermented Sauropus androgynus leaves to the diet is effective in reducing the content of fat or cholesterol and increasing the content of protein, iron, vitamin A and β-carotene of broiler meat. In addition, the compositions of amino acids and fatty acids of broiler meat are more balanced. Moreover, this product also improves the carcass quality without reducing body weight18 and the blood profile of broiler chickens19. The effectiveness of this product may be enhanced if the product is extracted because the extraction will better release the active compound. Therefore, the present study was designed to evaluate the effect of supplementation of fermented Sauropus androgynus leaf extract on amino acid and fatty acid compositions, cholesterol, protein, fat, iron and β-carotene of broiler meat.
MATERIALS AND METHODS
Extraction of fermented Sauropus androgynus leaves: Sauropus androgynus leaves were fermented by Saccharomyces cerevisiae17 and then they were extracted with hot water at 90°C for 20 min as described by Santoso et al.3. The contents of amino acids, fatty acids, protein, fat, iron, β-carotene and sterol were then measured.
Animals and experimental design: Two hundred broilers were purchased (strain Arbor Acres) from commercial hatcheries. On arrival, the broilers were placed in a single pen surrounded by a zinc ring and maintained on rice husks at a depth of approximately 5 cm. From 1-14 days of age, supplemental heat was provided using coal as fuel. The temperature was maintained at 32-34°C in the first week and gradually decreased in the second week. The broiler chickens were maintained on the floor in a house under continuous lighting. To avoid stress, the broiler chickens were immediately provided drinking water containing sugar and antistress. They were fed a commercial starter diet for 14 days.
At 15 days of age, the broiler chickens were weighed and selected based on body weight. A completely randomized design was used in the present study. The relative humidity and temperature of the house ranged from 65-75% and 23-32°C, respectively. A fan was turned on when the house temperature exceeded 29°C to prevent overheating. The experimental diets contained 19% crude protein and 3200 kcal ME kg–1. One hundred broiler chickens aged 15 days were distributed into five treatment groups. Each treatment group was represented by four replicates of five broiler chickens each. The 5 treatments were as follows: (1) Broiler chickens were fed a diet without fermented Sauropus androgynus leaf extract (FSALE) as the control (P0), (2) Broiler chickens were fed a diet supplemented with 4.5 g FSALE kg–1 diet (P1) (3) Broiler chickens were fed a diet supplemented with 9 g FSALE kg–1 diet (P2) (4) Broiler chickens were fed a diet supplemented with 13.5 g FSALE kg–1 diet (P3) and (5) Broiler chickens were fed a diet supplemented with 18 g FSALE kg–1 diet (P4). All broiler chickens were provided their diet and drinking water ad libitum.
Sampling and laboratory analysis: At the end of the study (aged 35 days), four broiler chickens for each treatment group were selected and slaughtered. The leg meat from each treatment was collected and analyzed for cholesterol, protein, fat, fatty acids, amino acids, iron and β-carotene. Fat and protein were determined by the method of AOAC20, whereas meat cholesterol was determined by the method described by Dinh et al.21,22. Amino acid composition was measured by the method as described by Henderson and Brooks23 and β-carotene was determined by the method of Grzelinska et al.24. Fatty acid composition of the meat was determined by the method described by De Almeida et al.25.
Data analysis: The experimental results were subjected to analysis of variance. Significant differences among the treatment groups were determined by Duncan's Multiple Range Test (DMRT) at p<0.05.
RESULTS AND DISCUSSION
Composition of FSALE: The components of fermented Sauropus androgynus leaf extract are presented in Table 1-3. The extract contained 25.46% protein, 1.34% fat, 3,642.6 μg β-carotene g–1, 8.17 mg iron g–1 and 0.17 mg sterol/100 mg. The extract was rich in palmitic acid (29.96%), glutamic acid (2.221%) and valine (0.523%). The contents of fat, iron and β-carotene of the extract were relatively similar to the contents of these nutrients in the leaf powder16. This means that the extraction did not increase the contents of these nutrients. The present study showed that FSALE contained a high sterol content.
Nutritional composition of broiler meat: The nutrient contents of FSALE are presented in Table 4. The results showed that FSALE inclusion significantly increased the contents of protein (p<0.01), β-carotene (p<0.001) and iron (p<0.001) of broiler meat, but it significantly reduced the contents of fat (p<0.05) and cholesterol (p<0.05) of broiler meat. The DMRT test showed that P0 had lower protein content than the other groups, whereas P0 had a higher fat content than P4, but it did not significantly differ from the other groups. Furthermore, P0 had lower β-carotene and iron contents than the other groups.
An increase in protein content of broiler meat indicated that there was improvement in the availability of protein for broilers because of better protein digestibilities. Chen et al.26 reported that soybean fermented by a Aspergillus and Lactobacillus mixture increased in vitro and in vivo protein digestibility. Adam et al.27 also found that fermentation increased in vitro protein digestibility.
As shown in Table 1, FSALE is rich in β-carotene and iron. Thus, FSALE might partly contribute to an increase in the β-carotene and iron contents of broiler meat. In addition, fermentation might improve the availability of β-carotene and iron for broilers. This result agreed with the observation of Santoso et al.17.
The compounds that play a role in lowering fat contents might be alkaloids and non alkaloids28, 3-O-β-D-glucosyl-(1→6)-β-D-glucosyl-kaempferol29, flavonoids30, tannins31 and polyphenol32.
Sterol found in FSALE (Table 1) may partly contribute to the lower cholesterol content as reported by Subekti4.
| Table 1: | Contents of protein, fat, β-carotene, iron and sterol of fermented Sauropus androgynus leaf extract |
| Table 2: | Composition of fatty acids of fermented Sauropus androgynus leaf extract |
| Table 3: | Composition of amino acids of fermented Sauropus androgynus leaf extract |
The other compounds that play a role in lowering cholesterol might be alkaloids and non-alkaloids28, saponins33, polyphenol32,34 and flavonoids35. Patil et al.36 reported that the reduction of cholesterol and triglycerides by alkaloid were in part caused by the reduction of lipogenic enzyme activities and increased bile acid excretion in feces.
Composition of fatty acids of broiler meat: The composition of fatty acids of broiler meat is presented in Table 5. The FSALE inclusion significantly reduced oleic acid (p<0.05) and increased docosahexaenoic acid (p<0.001) but had no effect on lauric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid and eicosapentaenoic acid (p>0.05). It was shown that P0 had lower docosahexaenoic acid than P2, P3 and P4 but was not significantly different from P1. The P0 had higher oleic acid than P1, P2 and P3 but was not significantly different from P4.
Docosahexaenoic acid is synthesized from a-linolenic acid37. The inclusion of linolenic acid from the extract may not be adequate to increase the synthesis of docosahexaenoic acid. Fermentation may enhance the digestion and availability of linolenic acid of FSALE resulting in higher linolenic acid as a substrate for docosahexaenoic acid synthesis. Table 5 shows that the linolenic acid contents of P1, P2 and P3 were lower than that of P0, suggesting that there is a conversion of linolenic acid to docosahexaenoic acid. Combining Lactobacillus sp. with flaxseed demonstrated the efficacy of conversion of α-linolenic acid to eicosapentaenoic acid and docosahexaenoic acid38. It was established that the inclusion of Sauropus androgynus leaf extract increased the number of Lactobacillus sp.39,40. Thus, an increase in Lactobacillus sp. may contribute to enhanced efficiency of linolenic acid conversion to docosahexaenoic acid.
The mechanism of lower oleic acid by the supplementation of FSALE is unknown. In poultry, linoleic acid and α-linolenic acid cannot be synthesized and tissue concentrations respond rapidly to dietary changes, whereas saturated and monounsaturated fatty acids, such as oleic acid, are synthesized and their concentrations are less readily influenced by diet41. However, Gallardo et al.42 reported that the oleic acid content of broiler meat was changed by diet.
Composition of amino acids of broiler meat: The composition of amino acids of broiler meat is presented in Table 6. The FSALE significantly reduced aspartic acid, serine, glycine, histidine, arginine, threonine and cystine (p<0.01), but FSALE significantly increased glutamic acid, alanine, proline, tyrosine, valine, methionine, phenylalanine and lysine (p<0.001).
It was shown that P0 had higher aspartic acid than P1 and P3. P3 had higher glutamic acid than the other groups. P1 and P3 had lower serine, glycine, histidine, threonine and cysteine than P0, P2 and P4. P1 had lower arginine than the others and P3 had lower arginine than P0, P2 and P4. The P1 and P3 had higher alanine, proline, tyrosine, valine, methionine, phenylalanine and lysine than the other groups. The extract had no effect on isoleucine and leucine.
The mechanism of an increase in lysine in P1 and P3 is still unknown. It has been established that lysine is an essential amino acid in animals, thus it is not synthesized in the body. Most bacteria synthesize lysine from aspartic acid. Sauropus androgynus leaf extract increased the number of Bacillus subtilis40and Lactobacillus sp.39,40. Thus, it is assumed that these bacteria may synthesize lysine from aspartic acid.
The low glutamic acid in P1 might cause lower arginine synthesis by bacteria and therefore resulting in lower arginine content in P1. Hood and Lyman43 and Xu et al.44 reported that glutamic acid has an important role in arginine synthesis.
Al-Fataftah et al.45 reported that an increase in Lactobacillus sp. enhanced the contents of lysine, methionine and cystine, as well as vitamin B12 and vitamin B6 in broiler meat.
| Table 4: | Effect of fermented Sauropus androgynus leaf extract on the nutritional composition of broiler leg meat |
P0: Broiler chickens were fed a diet without supplementation of fermented Sauropus androgynus leaf extract (FSALE) as the control, P1: Broiler chickens were fed a diet supplemented with 4.5 g FSALE kg–1, P2: Broiler chickens were fed a diet supplemented with 9 g FSALE kg–1, P3: Broiler chickens were fed a diet supplemented with 13.5 g FSALE kg–1, P4: Broiler chickens were fed a diet supplemented with 18.0 g FSALE kg–1. Values in the same row with different superscripts are significantly different. *p<0.05, **p<0.01, ***p<0.001 | |
| Table 5: | Effect of fermented Sauropus androgynus leaf extract on the fatty acids composition of broiler leg meat |
P0: Broiler chickens were fed a diet without supplementation of fermented Sauropus androgynus leaf extract (FSALE) as the control, P1: Broiler chickens were fed a diet supplemented with 4.5 g FSALE kg–1, P2: Broiler chickens were fed a diet supplemented with 9 g FSALE kg–1, P3: Broiler chickens were fed a diet supplemented with 13.5 g FSALE kg, P4: Broiler chickens were fed a diet supplemented with 18.0 g FSALE kg–1. Values in the same row with different superscripts are significantly different. *p<0.05, ***p<0.001, ns: Non significant | |
| Table 6: | Effect of fermented Sauropus androgynus leaf extract on the composition of amino acids of broiler leg meat |
P0: Broiler chickens were fed a diet without supplementation of fermented Sauropus androgynus leaf extract (FSALE) as the control, P1: Broiler chickens were fed a diet supplemented with 4.5 g FSALE kg–1, P2: Broiler chickens were fed a diet supplemented with 9 g FSALE kg–1, P3: Broiler chickens were fed a diet supplemented with 13.5 g FSALE kg–1, P4: Broiler chickens were fed a diet supplemented with 18.0 g FSALE kg–1. Values in the same row with different superscripts are significantly different. **p<0.01, ***p<0.001, ns: Non significant | |
They also reported that the microbial strains had the potential for enhancing biosynthesis of vitamin B12, vitamin B6, lysine, methionine and cystine.
An increase in Lactobacillus sp. in the gastrointestinal tract by Sauropus androgynus leaves39,40 might stimulate methionine synthesis and therefore the increased methionine content of broiler meat in P1 and P3. Al-Fataftah et al.45 reported that an increase in Lactobacillus sp. enhanced the contents of methionine. Homoserine is the common precursor for isoleucine, threonine and methionine. It was assumed that because of inadequate homoserine as substrate for methionine and threonine synthesis, poultry tend to synthesize methionine rather than threonine resulting in a lower threonine content. A lower glycine content might be partly caused by the conversion of glycine to serine46.
Creek47 reported that phenylalanine could be converted to tyrosine in poultry, whereas methionine could be converted to cystine. It appears that the conversion of methionine to cystine was inhibited resulting in higher methionine and lower cystine in P1 and P3. In addition, the higher conversion of phenylalanine to tyrosine may partly explain the higher tyrosine. Another possible mechanism of higher tyrosine is the lower conversion of tyrosine into dopamine, norepinephrine and epinephrine.
The contribution of glutamic acid from FSALE may not fully explain the higher glutamic acid in P3 because other treatment groups did not show this. Glutamic acid is synthesized from ammonia and α-ketoglutarate by the action of glutamate dehydrogenase. In contrast, α-ketoglutarate can be produced by oxidative decarboxylation of glutamic acid by glutamate dehydrogenase.
Glutamic acid can synthesize aspartic acid, alanine, proline and arginine. Thus, it appears that the glutamic acid in P1 and P3 may be inadequate for synthesis of aspartic acid resulting in lower aspartic acid in these groups compared with the control. The body may focus on synthesis of alanine and proline from glutamic acid resulting in higher levels of alanine and proline in P1 and P3.
Phenylalanine is produced by most microorganisms from prephenate48 in which this compound is decarboxylated with the loss of the hydroxyl group to produce phenylpyruvate. This compound is then transaminated using glutamate as the nitrogen source to produce phenylalanine and α-ketoglutarate. Amin and Onodera49 stated that phenylalanine is also synthesized from phenylacetate by microorganisms. Sauropus androgynus contains cis-2-methyl cyclopentanol acetate50, which may be converted to acetate and may be further converted to phenylacetate in the gastrointestinal tract by microorganisms.
The contribution of valine from Sauropus androgynus may partly explain higher valine contents in P1 and P3. A lower histidine content in meat may reduce the formation of carnosine, which may increase the lipid oxidation of meat. However, since alanine also has an important role in the synthesis of carnosine, the increase in lipid oxidation may be prevented. Kralik et al.51 reported that histidine and alanine inclusion in the chicken’s diet reduced lipid oxidation of chicken meat.
CONCLUSION
Fermented Sauropus androgynus leaf extract (FSALE) contained 25.46% protein, 1.34% fat, 3,642.6 μg β-carotene g–1, 8.17 mg iron g–1 and 0.17 mg sterol/100 mg. The extract was rich in palmitic acid (29.96%), glutamic acid (2.221%) and valine (0.523%). The FSALE inclusion at a level of 4.5-18 g kg–1 in the diet increased the contents of iron, protein and β-carotene but it reduced the content of cholesterol of broiler meat. In addition, FSALE inclusion improved the compositions of amino acids and fatty acids.
SIGNIFICANT STATEMENT
This study discovered the possible uses of fermented Sauropus androgynus leaf extract, which can be beneficial for enriching nutrients such as protein, iron and β-carotene with lower fat and cholesterol in broiler meat. This study will help to uncover the critical area of high fat deposition but low protein, iron and β-carotene deposition in broiler meat that many researchers were not able to explore previously. Thus, a new theory may be created based on the usefulness of fermented Sauropus androgynus extracts on lowering fat deposition and enriching protein, iron and β-carotene in broiler meat.
ACKNOWLEDGMENT
This study was supported by funding from the Directorate General of Higher Education, Ministry of Research, Technology and Higher Education under contract number 044/SP2H/LT/DRPM/2016.