BIOTROPIA Vol. 31 No. 1, 2024: 44 - 53  |  DOI: 10.11598/btb.2024.31.1.2030

EFFECT OF BOILING CARICA PUBESCENS SEEDS ON ANTINUTRIENT CONTENT AND IN VITRO NUTRIENT DIGESTIBILITY


RAHMA WULAN IDAYANTI1,2*, TATIK ROHANI 2, FENDI YAYUKI 2, ALFIAN NUR SYAHRUL2, AJENG AYU SAP UTRI2, ENDANG PUJI LESTARI,2AGUN PUJI LESTARI 2agunG PurnOmOaDI 1, ENDANG PURBOWATI 1AND MUKH ARIFIN 1

1Department of Animal Science, Faculty of Animal and Agricultural Sciences, Diponegoro University, Semarang 50275, Central Java Indonesia
2Department of Animal Science, Faculty of Agriculture, Tidar University, Magelang 56116, Central Java Indonesia

*Corresponding author, e-mail: rahmafina2@gmail.com

Received 29 May 2023 / Revised 30 November 2023 / Accepted 4 January 2024


ABSTRACT

This research was conducted to determine the chemical composition and anti-nutritional content of Carica Dieng seeds (Carica pubescens) and their in vitro digestibility after boiling. The process of boiling is frequently employed to reduce antinutrient content and increase the digestibility of nutrients. This research used a completely randomized design, consisting of three treatments with boiling durations of 0, 10, and 20 minutes, each repeated six times. The research revealed a significant (p<0.05) decrease in the tannin and saponin levels of Carica Dieng seeds following the boiling process. The concentration of carica seed tannin was lowered to a range of 36-48%, while the saponin content decreased by 52-58%. Subsequently, a descriptive analysis was conducted to assess the nutritional and amino acid composition of the seeds after boiling them for a duration of 10 minutes. Lysine (1.57% w/w) dominated the essential amino acids in Carica pubescens seeds, and glutamic acid (3.19% w/w) dominated the non-essential amino acids. The digestibility of boiling carica seeds as feed was assessed by including them into complete feed at different proportions: 0% (T0), 5% (T2), 10% (T3), and 15% (T4). This experiment followed a Completely Randomized Design with five replications. The variables included in-vitro dry matter (DMD) and organic (OMD) digestibility. The DMD of carica seeds at a concentration of 0% showed a statistically significant difference (p<0.05) when compared to the addition of 5% carica seeds. However, there was no significant difference in DMD between adding carica seeds at levels of 10% and 15% in the total meal. The organic material digestibility variable showed that the treatment without carica seeds was not significantly different from adding 10% carica seeds but was substantially different from adding 5 and 15% carica seeds. Carica pubescens seed supplementation of up to 10-15% remains viable for inclusion in whole feed since it does not reduce digestibility.


Keywords: boiling, carica pubescens, , in-vitro digestibility, saponin, tannin



INTRODUCTION

The feed factor is an essential element of a ruminant fattening strategy. The availability and synergistic interaction of nutrients in the diet are crucial factors in determining the productivity of animals. Alternative feed sources are necessary due to the typically expensive cost commercial feed and the intermittent availability of protein feed. Consequently, meeting the nutritional requirements of cattle and maintaining a consistent feed supply can be labor-intensive. Agro-industrial waste can serve as a potential substitute for cattle feed due to its retention of essential nutrients and consistent availability. The addition of agro-industrial wastes, such as carica processed waste, to complete feed is expected to minimize feed costs, reduce waste, and increase small-scale animal production (Lalramhlimi et al. 2022). One of the initiatives to build a low-cost and effective feed business is using locally accessible agricultural and plantation wastes in each region, along with reducing agro-industrial waste. Despite the extensive utilization of the country's abundant feed ingredients, the need for these feeds remains unfulfilled (Kim et al. 2019). Therefore, further research is required to investigate unconventional materials. Carica Dieng confections have become into basic commodities, possessing economic significance. Annually, a total of 1100-1200 tons of fruit is harvested, with the involvement of 30 middle, small, and micro businesses (Ningsih et al. 2019).

The Carica seeds and their membranes are remnants resulting from the preparation of the carica fruit. According to Briones-Labarca et al. (2015), the ripe seeds have a moisture content of 3.50%, an ash content of 3.96%, a crude protein content of 31.84%, and a crude fiber content of 24.41%. The relatively high protein content of Carica Dieng seeds might provide some advantage. Multiple research studies have proven that Dieng carica seeds contain anti-nutritional factor tannins and saponins (Kiranmayi 2014); hence, the utilization of Dieng carica seeds as feed needs further research. Natural foods and feedstuffs contain chemical substances known as anti-nutritional factors (ANFs) which are generated through the metabolic processes of species and various factors that hinder optimal nutrition. These factors include the inactivation of particular nutrients, a deceleration of the digestive system, and a reduction in the metabolic utilization of the food or feed (Thakur et al. 2017). Tannins are polyphenolic chemicals classified into two types depending on their chemical structure: condensed and hydrolyzable tannins. Green plants typically produce different quantities of tannins, resulting in different biological properties. The presence of many phenolic groups accounts for tannins' strong affinity for proteins. These provide numerous opportunities for peptide carbonyl groups to bind. The creation of such complexes is specific regarding the tannin and protein involved, with the degree of affinity between the molecules in each chemical property (Bunglavan and Dutta 2013). According to Trisnadewi et al. (2014), an increased concentration of tannins attaches to carbohydrates and proteins, thereby impeding the ability of rumen microorganisms to degrade these substances and consequently diminishing enzyme activity. A reduction in the population of rumen bacteria will correspondingly lead to impaired nutrient assimilation and decomposition, as well as reduced access to carbohydrates and proteins within the rumen.

Saponin is a glycoside that has an aglycone in the form of sapogenin. The chemical structure of saponins is in the form of a glycoside consisting of glycone and aglycones. The glycone part is a sugar group such as glucose, fructose, and other types of sugar, while the aglycone part is a sapogenin (Nurzaman et al. 2018). Saponins are glycosides with aglycones in the form of steroids and triterpenoids (Yanuartono et al. 2017). Steroid saponins are often found in single-seeded or monocot plants (Negi et al. 2013), and triterpenoid saponins are often found in double-seeded plants or dicots (Yanuartono et al. 2017). Anti-nutritional factors (ANFs), which are naturally present compounds in plants, have the potential to adversely affect the value of feed when administered to livestock in specific amounts. They can affect biological aspects, disrupt body metabolism, reduce livestock productivity, and inhibit livestock growth and health (Jayanegara 2018; Murni et al. 2012). Because ANFs can stunt the growth of livestock that consume them, their presence in feed ingredients may be a ration-limiting factor. It is necessary to study the ANFs content in Carica Dieng seeds. This step is critical to minimize the various adverse effects of ANFs. Boiling has been identified as a potential technique for diminishing the antinutrient content (Ndidi et al. 2014). In their research, Assam et al. (2019) demonstrate that water steaming can also reduce the content of tannins, HCN, saponins, and phytate.

The ability of ruminant livestock to utilize feed containing antinutrients varies. The tolerance of sheep to the antinutrients in boiled carica seeds has yet to be discovered, thus requiring research on the levels of carica seeds in sheep rations. The addition of boiled carica seeds at different levels aims to determine the maximum limit of antinutrients that sheep can tolerate. With this knowledge, maximizing the use of carica seeds, which are processed by boiling as a source of protein in sheep, can be used as safe feed for their productivity. It is necessary to do in vitro testing on boiled carica seeds to assess the nutrient quality, specifically the digestibility of dry matter and organic matter in sheep rations. It can be achieved by replicating the physiological processes that occur in the digestive system of livestock.


MATERIALS AND METHODS

Time and Location of Research

The Dieng carica seeds used in this study were collected from the carica beverage manufacturing company Dieng, Banjarnegara, Central Java, Indonesia. The collection of seeds was conducted in April 2022 (Figure 1). The Carica Dieng fruit and its seeds were used as samples in this study (Figure 2).

Procedures

The carica seeds were separated from the carica fruit through peeling and removal. The seeds underwent processing in accordance with the procedure outlined by Talabi et al. (2016). The boiling process lasted for durations of 0, 10, and 20 minutes. In each case, the water was heated until it reached the boiling point prior to pouring the seeds, and they were then left to boil for the duration length. Subsequently, the seeds were drained of water, dried, and boiled. They were then examined to determine the levels of tannins, saponins, amino acids, as well as the digestibility of dry and organic matter using in vitro analysis.

Nutritional Component Analysis

Chemical analysis of carica seed samples that had been boiled included proximate analysis: dry matter (DM), ash, organic matter (OM), crude protein (CP), ether extract (EE), and crude fiber (CF), according to AOAC (2005); Amino acid analysis In house method (ICI Instrument Method 1988), tannin content (Folin Ciocalteau method (Chaovanalikit and Wrolstad, 2004), saponins (Pasaribu et al. 2014), dry and organic matter digestibility was determined using the Tilley and Terry method (1963).

Figure 1: Location of the survey (Dieng, Banjarnegara District Central Java Province, Indonesia)


a

b

Figure 2: a. Carica pubescens fruit; b. Carica fruit flesh; c. Carica pubescens seeds

c

table 1: Feed composition and nutritional content of the diets.


Experimental Design and Data Analysis

Carica Dieng seeds used in this research were collected from Dieng, Central Java, Indonesia. As part of the preliminary investigation, the experiment examined the existence of tannins and saponins and assessed the reduction of their levels by a physical treatment (boiling). This research used a completely randomized design consisting of three treatments: T0 without boiling, T1 = 10 minutes, T2 = 20 minutes, and six replications. The Carica seeds were subjected to boiling water for an appropriate duration during their processing. Their amino acid composition and the best results were carefully examined and discussed descriptively. Carica seeds were added to complete feed and tested in vitro to evaluate the digestibility of dry matter and organic matter. Testing was done using a completely randomized design with four treatments and five replications. Carica seeds were added to complete feed, consisting of T0 = 40% Pak Chong grass + 60% concentrate + 0% addition of carica seeds, T1 = 40% Pak Chong grass + 55% concentrate + addition of 5% carica seeds, T2 = 40% Pakchong grass + 50% Concentrate+ addition of 10% carica seeds, T3 = 40% Pakchong Grass+45% Concentrate+ addition of 15% carica seeds (Table 1). Data on the presence of tannins and saponins and their digestibility were analyzed using analysis of variance (ANOVA) at the 5% level. If there were differences between treatments, continued further testing with the Duncan Multiple Range Test (DMRT).

Experimental Design and Data Analysis

Carica Dieng seeds used in this research were collected from Dieng, Central Java, Indonesia. As part of the preliminary investigation, the experiment examined the existence of tannins and saponins and assessed the reduction of their levels by a physical treatment (boiling). This research used a completely randomized design consisting of three treatments: T0 without boiling, T1 = 10 minutes, T2 = 20 minutes, and six replications. The Carica seeds were subjected to boiling water for an appropriate duration during their processing. Their amino acid composition and the best results were carefully examined and discussed descriptively. Carica seeds were added to complete feed and tested in vitro to evaluate the digestibility of dry matter and organic matter. Testing was done using a completely randomized design with four treatments and five replications. Carica seeds were added to complete feed, consisting of T0 = 40% Pak Chong grass + 60% concentrate + 0% addition of carica seeds, T1 = 40% Pak Chong grass + 55% concentrate + addition of 5% carica seeds, T2 = 40% Pakchong grass + 50% Concentrate+ addition of 10% carica seeds, T3 = 40% Pakchong Grass+45% Concentrate+ addition of 15% carica seeds (Table 1). Data on the presence of tannins and saponins and their digestibility were analyzed using analysis of variance (ANOVA) at the 5% level. If there were differences between treatments, continued further testing with the Duncan Multiple Range Test (DMRT).


RESULTS AND DISCUSSION

Effect of Processing Antinutritional (Tannin, Saponin) Content

This research shows that there is an influence between the boiling process on reducing the tannin and saponin levels of Carica Dieng seeds (p<0.05), where the boiling treatment at T2 and T3 significantly decreased compared to T1, but T2 and T3 were relatively the same. Anti-nutritional quality of Carica pubescens seeds with prolonged boiling treatment is shown in Table 2. The tannin content of the T2 treatment (boiling for 10 minutes) was 2.67% lower than the T1 treatment (raw materials without boiling). The tannin concentration decreased to (2.21%) under T3 treatment (20 minutes of boiling). This study relied on the research conducted by Jamarun et al. (2021), who find that mangrove tannin levels fall by 7.4% after 10 minutes of boiling. Assam et al. (2019) report that boiling for up to 30 minutes reduce the tannin and saponin content in Cassia tora seeds. This drop happened as a result of the lengthy boiling time, which might cause the connections between the fibers in carica seeds to loosen. Due to the osmosis process, which pushes tannins out of the leaves and causes them to dissolve in alkaline water through diffusion activity, the tannin content will be reduced (Perdana et al. 2012). Acids, bases, and enzymes can hydrolyze tannin. Tannins will break down into glucose and gallic acid if heated at 98.89ºC - 101.67ºC (Muhammad et al. 2015). Tannins are readily soluble in water, and solubility increases when dissolved in hot water (Perdana et al. 2012). According to the theory that heat denatures proteins and because some antinutritional components present in carica seeds are protein compounds, heat tends to denature them, the reduction in antinutrient levels caused by boiling in this study. While tannins discolor seeds and bind protein through hydrogen bonds and hydrophobic interactions.

Table 2 indicates that the saponin content of Carica pubescens seeds decreased from 21.23% to 8.86%. This result is consistent with a previous study by Lakram et al. (2018). They report that boiling for 25 minutes at 100 ˚C significantly reduce saponin levels. The reduction in saponin content can be attributed to the thermal degradation associated with the heat supplied to the cooked plants. The decrease was also observed by Hemmige et al. (2017) and Ilelaboye et al. (2013). Saponins are water-soluble chemicals that are highly soluble in cold and hot water (Chairunnisa et al. 2019).

Table 2: Antinutritional Content (Tannin and Saponin) of Carica pubescens seeds
Antinutritional Content (Tannin and Saponin) of Carica pubescens seeds
Note: Different superscripts denote significant differences (P<0.05) within the same column.

Saponin is a complex glycoside that is found in plants, known for its foaming properties. Therefore, it will produce foam when it reacts with water and is shaken. Boiling may also remove components from the material that are deemed superfluous, particularly those that are water-soluble. Temitope et al. (2013) identify that heating can reduce saponin concentrations in various plant species. According to Chaturvedi et al. (2012), boiling can lower the amount of saponin in soybeans (Glycine max Linn). Saponins and plants with high saponin content harm protozoa by building an irreversible combination with steroids in the protozoan cells (Yanuartono et al. 2017). This decrease in the protozoa population in the rumen is likely to have several beneficial effects, including improved nitrogen metabolism efficiency, decreased methane gas emissions, changes in the population of bacteria and fungi in the rumen, and the potential for increased flow of bacterial protein to the lower digestive tract. The findings of this study were corroborated by Wang et al. (2012), who claimed that adding saponins from tea to the diet might suppress methanogenesis. This reduction is expected to have positive effects on the environment and enhance the productivity of animal rearing for feed purposes.

Chemical Composition

The results of boiling the seeds with the time of 10 minutes were tested descriptively for their nutrient content and amino acids. The proximate analysis showed that carica seeds contained 12.75% Moisture, 6.57% Ash, 14.85% Fat, 27.98% Crude Protein, and 29.89% Crude Fiber, 35.32% Nitrogen Free Extract (Table 3). More than 20% of the protein in Carica pubescens seeds makes them an excellent source of protein for rations. High molecular weight peptides make up protein, a chemical material with various bodily functions. The primary building block of muscle tissue is protein.

The amount of microbial protein supply, which is dependent on the availability of carbohydrates and nitrogen (not always in the form of protein), as well as the amount of protein that is freely degraded in the rumen, is the primary determinant of the amount of essential and non-essential amino acids that are available to tissues in ruminants (Wu et al. 2014). The optimal protein requirement for ruminants is determined by their capacity to digest protein. Concentrate feed containing a variety of proteins can provide a more cost-effective source of protein. It is because the combination of proteins with multiple types and amounts of amino acids increases the ability of rumen microbes to rapidly synthesize all the necessary amino acids (Williamson and Payne, 1978; Pathak, 2008).

Table 3: Chemical composition of Carica pubescens seeds (% DM)
Table 3 Chemical composition of Carica pubescens seeds (% DM)
Note: a)proximate analysis (DM: dry matter, CP: crude protein, CF: crude fiber, Drug and Animal Testing Laboratory, Veterinary Agency Semarang District (2022). b) ash, EE: ether extract, Laboratory of Animal Nutrition Faculty of Animal and Agricultural Sciences, Diponegoro University (2022). NFE: nitrogen-free extract

Table 4 presents the results of the analysis of amino acids from the seeds of Carica pubescens. Carica pubescens seed essential amino acids are dominated by lysine (1.57% w/w), and non-essential amino acids by glutamic acid (3.19% w/w). Amino acids, which are found in proteins, serve the purpose of constructing new tissues, facilitating energy metabolism, synthesizing hormones, and acting as essential catalysts for several physiological processes in the body (Li et al. 2020).

Table 4: Amino Acid Composition of Carica Pubescens seeds (% w/w)
Table 4 Amino Acid Composition of Carica Pubescens seeds (% w/w)
Note: Unit Laboratorium Jasa pengujian, Kalibrasi dan Sertifikasi Institus pertanian Bogor University, 2022.

Carica pubescens seeds have a relatively high protein content, which is considered important as a new source of nutrition for ruminants. The protein content in Carica pubescens seeds is around 24%, equivalent to that of coconut meal. Amino acid analysis using HPLC showed that the seeds of Carica pubecens contained essential and non-essential amino acids (Table 2). The highest essential amino acid from Carica pubescens seeds is dominated by lysine (1.57% w/w). Lysine is necessary because it is one of the precursors to the growth of rumen microorganisms (Kong et al. 2021). Lysine has a part in forming carnitine, which can stimulate growth, guard against ammonia toxicity, and improve the body's ability to fend off extreme temperature fluctuations (Aristasari et al. 2018). Lysine inclusion in the meal can promote increased protein synthesis. Feed efficiency and feed consumption can both be improved by lysine content (Aristasari et al. 2018). Lysine tends to boost the feed's protein, fat, and calorie content when added to the ration. The energy content of the meal can also determine the level of feed efficiency. Beyond that, Lysine can improve a feed's ability to be digested by the ileum (intestine). Livestock may be absorbed fast to increase feed efficiency and give livestock a feeling of fullness and strong growth rates. The amino acid tyrosine, which regulates the body's reaction to stress, can be more easily digested after lysine has been added to feed increases the anti-inflammatory action and aids in the healing and prevention of ulcerative colitis and peptic ulcers (Aristasari et al. 2018).

The highest non-essential amino acids from Carica pubescens seeds were glutamic acid content (3.19% w/w). The creation of proteins depends on glutamic acid, which also serves as a source of energy for the intestinal lining cells. Enhances immunological response. Glutamic acid is a non-essential amino acid that functions as a building block for protein, as a precursor for several non-essential amino acids, and helps the body's metabolism (Slyamova et al. 2016) as well as a neurotransmitter for taste (Huang et al. 2019). Glutamic acid also functions as an antioxidant regulating inducible nitric oxide synthase (iNOS) as protection against intracellular parasites, bacteria, fungi, viruses, and protozoa (Maslami et al. 2018). Glutamic acid is an alternative feed additive that is safe and environmentally friendly in improving carcass performance and quality (Maslami et al. 2018).

In-vitro dry matter and organic matter digestibility

Digestibility is an initial sign of the availability of different nutrients in a food source that livestock will eat. The high nutritional content consumed post-digestion indicates that the meal is easily digestible (Mayulu et al. 2019). Table 5 shows the digestibility of dry and organic matter with the addition of carica seed content. The T0 treatment, which did not involve the addition of carica seeds, showed no significant difference compared to the treatments with 10% and 15% carica seeds. However, it exhibited a major difference in dry matter digestibility compared to the treatment with 5% carica seeds. The study found that the lowest digestibility was observed when employing a 5% concentration of Carica. This is believed to be due to the varying levels of Carica pubescens seeds used in the study, which resulted in diverse digestibility outcomes. Carica levels cause different nutrient content, such as the amount of crude protein, crude fiber, and tannins in the treated feed. This decrease in digestibility is likely caused by increased crude fiber content due to adding Carica pubescens seeds to complete feed. Crude fiber is a component of organic material that is difficult to digest in rumen. Increasing the crude fiber content will reduce the digestibility of dry matter, crude protein and energy. Setiasih et al. (2023) explained that in their research, the more crude fiber in the feed material, the thicker the cell walls and the more resistant it is to fiber-digesting microorganisms, causing the digestibility of the material to be lower. Rumen microorganisms participate in the fermentation process. Feed intake depends on activity, and the nutrients contained in feed ingredients impact rumen microbial activity (Sandi et al. 2015). An increase or decrease in digestibility is shown in the number and activity of microbes (Mayulu 2014). The presence of tannins in carica seeds is thought to reduce the digestibility of feed ingredients. This decrease was due to tannin being able to bind protein so that rumen microbes could not degrade it. However, if the tannin concentration is not well controlled, the presence of tannins in protein protection will enhance the availability of high-quality feed protein. This can lead to disruptions in overall digestibility and nutrient absorption, since tannins interact with protein, fiber, vitamins, and minerals. In addition, Mertens & Grant (2020) state that the physical composition of feed ingredients and their ratios vary. Composition, temperature and speed through the digestive tract are some variables that influence the digestibility of dry and organic matter.

The study examined the impact of Carica seed content on the digestibility of organic matter. The treatment without any carica seed addition showed no significant difference compared to the treatment with 10% carica seed. However, it did show a significant difference when compared to the treatments with 5% and 15% carica seed content. The digestibility of organic matter describes the availability of nutrients from feed. The livestock digestive system can digest organic materials, which include food in the form of organic material components, including carbohydrates, proteins, lipids and vitamins. In order to make organic components in feed more accessible, it is essential to convert them from insoluble to soluble form (Suardin et al. 2014). Ismail (2023) assert that the digestibility of organic matter is closely related to the digestibility of dry matter because some dry matter consists of organic matter. Factors that influence the digestibility of organic materials are the crude fiber. The increasing crude fiber content in feed tends to increase the cellulose, hemicellulose and lignin content, is caused by microbes being unable to optimally digest the crude fiber components contained in feed ingredients resulting in lower organic matter digestibility values (Tafsin 2019).

Table 5: In-vitro dry matter (DM) and organic matter (OM) digestibility of Carica pubescens seeds
Table 5 In-vitro dry matter (DM) and organic matter (OM) digestibility of Carica pubescens seeds
Note: Different superscripts denote significant differences (P<0.05) within the same column.


CONCLUSION

The research findings indicated a significant decrease in the antinutrient content (tannins and saponins) of boiling Carica pubescens seeds. The inclusion of Carica seed levels in complete feed ingredients can meet the protein requirements as they have a high protein content. Adding 10-15% of Carica seed levels does not negatively affect in vitro digestibility.


ACKNOWLEDGMENTS

This work was partially supported by Puslapdik Kemdikbud (Center for Education Financial Services, Ministry of Education, Cultural Research and Technology, LPDP (Indonesian Education Endowment Fund) to fund this research through the Indonesian Education Scholarship (BPI) or the Indonesian Education Scholarship Program, the Faculty of Animal Husbandry and Agriculture at Diponegoro University, and the Faculty of Agriculture at Tidar University.


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