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Study of nyamplung (Calophyllum inophyllum) kernel cake as an alternative protein source for ruminant feed and its effect on methane emission through in vitro

Dimas Hand Vidya Paradhipta, Chusnul Hanim, Ali Agus, Budi Leksono1, Aziz Umroni1, Sinta Maharani2, Arrynda Rachma Dyasti Wardani and Moh Sofiul Anam

Department of Animal Nutrition and Feed Science, Faculty of Animal Science, Universitas Gadjah Mada, Yogyakarta 55281, Indonesia
1 Research Center for Plant Conservation, Botanic Gardens, and Forestry, National Research and Innovation Agency (BRIN), Cibinong 16911, Indonesia
2 Research Center for Applied Zoology, National Research and Innovation Agency (BRIN), Cibinong 16911, Indonesia


The industry of Nyamplung/tamanu (Calophyllum inophyllum) crude oil is developed massively in Indonesia. The by product of this crude oil is called nyamplung kernel cake (NKC). This study was conducted to investigate the chemical compositions, plant secondary metabolites, and in vitro digestibility of NKC as new alternative feed. In chemical compositions, the NKC contained 90.2% dry matter, 7.92% organic matter, 20.1% crude protein, 15.3% ether extract, 53.3% neutral detergent fiber, 38.2% acid detergent fiber, and 15.1% hemicellulose. In plant secondary metabolites, the NKC contained 6.47% total phenol, 1.70% total flavonoid, and 0.90% saponin. The total tannin of NKC was 0.93% consisting of condense tannin at 0.45% and hydrolysable tannin at 0.48%. The chemical compositions of NKC were similar to palm kernel cake (PKC), copra cake (CC), tamarind (Leucaena leucocephala) leaves meal (TLM), gliricida (Glircidia sepium) leaves meal (GLM), and malapari (Pongamia pinnata) kernel cake (MKC). Thus, those feedstuffs were incubated in rumen buffer for 48 h. During ruminal incubation, NKC produced the lowest methane emission without any negative effects on rumen fermentation indices. Moreover, NKC still had a similar digestibility to PKC, CC, and GLM. This study concluded that the NKC can substitute the use of PKC, CC, TLM, GLM, and MKC in the ruminant diet, which can also help to reduce methane emission through in vitro study.

Keywords: Calophyllum inophyllum, feed, in vitro, methane, nyamplung kernel cake, protein source


Nyamplung/tamanu (Calophyllum inophyllum) is an origin plant of Indonesia and speared from Sumatra to Papua islands (Leksono et al, 2014). Nyamplung plant produce a kernel, which can be used to make tamanu crude oil (TCO) as an alternative of biofuel. The kernel is extracted using pressing method to result TCO. Biofuel from TCO was reported to result in lower emission of carbon monoxide compared to commercial biofuel. In addition, TCO have also been used for cosmetic and herbal medicine in Indonesia (Leksono et al, 2014). Due to a high benefit and purpose, the industry of TCO in Indonesia is developed with widely market. Nowadays, there are 3 manufactures that produce TCO to supply a market demand in Central Java Province, Indonesia. Moreover, the nyamplung kernel produce higher crude oil compared to castor ( Jatropha curcas L.), candlenut (Reutealis trisperma), and similar production with palm kernel (Elaeis guineensis) (Leksono et al, 2016). In general, nyamplung can produce 11.7 kg crude oil per tree(Leksono et al, 2014; Leksono et al, 2016).

The industry of TCO results a waste, called nyamplung kernel cake (NKC), which is a by-product after extraction to get TCO. Extraction process of nyamplung kernel results a TCO and a NKC at ratio 1:1. This indicated that the production of NKC as waste product is high and will increase following the demand of TCO in the market. In the field, the NKC already applied to the ruminant by local farmers. Previous study reported that NKC has a crude protein (CP) around 21.7% - 23.6% (Leksono et al, 2017), which has a high potential as alternative unconventional feed for animal feed. However, another study on NKC did not reported yet. As a waste product from TCO, the NKC may contain a secondary metabolite, which may be anti-nutritional factor or can help to reduce a methane (CH4) emission when applied as ruminant feed. Nyamplung kernel contains saponin, alkaloid, flavonoid, and tannin (Udarno and Tjahjana, 2019) that can remain in the NKC. In addition, NKC can be used as sources of protein with also providing a plant secondary metabolite as rumen modifier to reduce a methane emission (Jayanegara et al, 2010; Bodas et al, 2012). Almost the feedstuffs of protein source have a high price, which are unaffordable for local farmers. As a by-product, the NKC has a cheap price with sustainable production, which can be developed as new alternative protein sources. Therefore, the present study was aimed to investigate the chemical compositions, plant secondary metabolites, and in vitro digestibility of NKC as new alternative feed. After, the NKC was compared with several feedstuffs, which have similar chemical compositions, to evaluate the digestibility, fermentation indices and methane production in the rumen through in vitro study. The present study was an initiation research to develop unconventional feedstuff, which can help to reduce a methane emission. The results of the present study can be used as a reference to evaluate the potency of NKC as new ruminant feed in Indonesia.

Material and method

Chemical Compositions and plant secondary metabolites

Total of 30 kg NKC was composited from 3 batches of TCO extraction and collected from Purworejo Distric, Central Java Province. The pictures of nyamplung kernel and the NKC are presented in Photo 1. The NKC was sub-sampled approximately at 1 kg for chemical compositions analysis. The sub-sampled NKC was placed into dry oven (Memmert UN55, Germany) at 55oC for 48 h. Dried samples were ground using a Wiley mill with 1 mm screen. For measuring dry matter (DM), total of 5 g of sample were dried at 105oC for 24 h (method 934.01 of AOAC, 2005). The organic matter (OM) was determined with a muffle furnace (Advantec KM-420, Japan) at 550oC for 5 h. The procedure of Kjeldahl (method 984.13 of AOAC, 2005) was used to determine crude protein (CP) using a N analyzer (B-324, 412, 435 and 719 S Titrino, BUCHI, Flawil, Switzerland). The procedure of Soxhlet (method 920.39 of of AOAC, 2005) was used to determine ether extract (EE). The neutral detergent fiber (NDF) and acid detergent fiber (ADF) were determined by using an Ankom 200 fiber analyzer (Ankom Technology, Macedon, NY, USA) according to the protocol of AOAC (AOAC, 2005; method 2002.04 and method 973.18, respectively). In addition, heat stable amylase was applied in NDF analysis. The hemicellulose (HEMI) was determined by calculating the differences between NDF and ADF.

Photo 1. Nyamplung kernel (A) and nyamplung kernel cake after oil extraction (B).

The secondary metabolites such as phenol, flavonoid, saponins and tannins were conducted using Spectrophotometry UV-vis (UV-1800 Shimadzu, Japan). The analysis of saponins was conducted based on the procedure of Pramono (2005), while the analysis of total phenol was conducted following Chaovanalikit and Wrolstad (2004). The determination of flavonoid used the procedure of Gonzalez and Herrador (2007). The analysis of hydrolysable and condensed tannin followed the procedure of Makkar et al (1993).


Total of 30 thin tailed male sheep were used to evaluate the acceptability of NKC. The sheep was chosen due to have a higher selectivity of feed than cattle. The acceptability test in the present study was conducted directly without any adaptation period to evaluate the fondness of NKC as a new feedstuff for ruminant. The sheep was placed in pen, which each pen contained 4 animals. The sheep had a random body weight (25 – 35 kg) and age (8 – 15 mo). Total of 20 g of single NKC was fed for each sheep at 1 pm during 3 day. The NKC did not fed to the animal at morning or at afternoon to reduce the effect of starving. The data was presented as the number of animals, which consumed the NKC totally per day.

Ruminal in vitro incubation

The NKC was incubated in rumen condition through in vitro method with several feedstuffs consisting of palm kernel cake (PKC), copra cake (CC), tamarind (Leucaena leucocephala) leaves meal (TLM), gliricida (Glircidia sepium) leaves meal (GLM), and malapari (Pongamia pinnata) kernel cake (MKC). Those feedstuffs contain high protein, which is relative similar to NKC. In addition, those feedstuffs also widely applied for local farmers in Indonesia. The PKC, CC, TLM was collected from local farmers, which had already prepared as dried powder. In addition, MKC was collected from Research Center for Plant Conservation, National Research and Innovation Agency (BRIN). All feedstuffs consisting of PKC, CC, TLM, GLM, and MKC were analyzed chemical compositions and results are presented in Table 1.

Table 1. Chemical compositions of feedstuffs in the present study (%, DM)








Dry matter






Organic matter






Crude protein






Ether extract






Neutral detergent fiber






Acid detergent fiber












1PKC, palm kernel cake; CC, copra cake; TLM, tamarind leaves meal; GLM, gliricida leaves meal; MKC, malapari kernel cake. DM, dry matter

In the preparation of rumen buffer, two cannulated Bali cattle (Bos indicus) was used as donor of rumen fluid. The rumen fluid was collected before morning feeding. The Bali cattle weighting in average 300 kg were fed rice straw and commercial concentrate mixed at a 7:3 ratio (CP 12% and ME 10 kcal/kg). Two layers of cheesecloth was prepared to filter a collected rumen fluid. The rumen buffer was made by mixing a rumen fluid with buffer solution at ratio 1:4 according to Tilley and Terry (1963). The buffer solutions was made following the procedure of McDougall (1948). Rumen buffer was gassed with carbon dioxide (CO2) to maintain an anaerobic condition (Paradhipta et al, 2023). Ruminal incubation was conducted into 100 mL glass serum bottle containing 500 mg dried sample and 40 mL of rumen buffer (Tilley and Terry, 1963). The glass serum bottle was closed tightly using rubber cap and aluminum sealer. Each treatment used quadruplicate using 4 bottles along with four blanks. Incubation was conducted at 39oC for 48 h in the aerobic incubator. After 48 h of incubation, 10 mL gas was collected from each bottle using syringe. The gas was transfered into vacuum tube for storage before methane analysis. Methane concertation were measured by gas chromatography (Shimidzu GC-2010) with an RT-OBond 30m capillary column, 0.32 nmID, 10µm DF, thermal conductivity detector (TCD). The carrier gas used helium gas with a flow rate of 1.5 ml/minute at a temperature of 50°C and a split ratio of 20. The TCD temperature was operated at 200°C with a current of 40 mA and the injector temperature is operated at 100°C. The concentration of methane was expressed as ppm.

The glass wool was prepared into gooch crucible to separate a sample from rumen buffer. After gas collected, all bottles were opened and then filtered using gooch crucible. The filtered sample was used to calculate the in vitro dry matter digestibility (IVDMD) and in vitro organic matter digestibility (IVOMD). The rumen buffer was analyzed pH using a pH meter (Ohaus AB23PH-F, China) and volatile fatty acid (VFA) using gas chromatography (GC 8A, Shimadzu Crop., Japan) according to the procedure of Hidayah et al (2023). The ammonia-N was determined using from rumen buffer using the colorimetric method of Chaney and Marbach (1962).

Ethical approval

The animal care and procedure for in vitro study was approved by The Animal Care and Use Committee, Faculty of Veterinary Medicine, Universitas Gadjah Mada (No 025/EC-FKH/Eks./2023)

Statistical analysis

All data of ruminal incubation were analyzed using completely randomized design using PROC GLM of SAS (SAS, 2002). The model was Y ij = µ + T i + eij, where Yijis response variable, µ is overall mean, Ti is the effect of feedstuffs, eij is error mean. The mean separation was conducted using Tukey test and the significant differences were declared at p ≤0.05. The data of chemical compositions and acceptability test of NKC were explored as descriptive.


Chemical compositions and plant secondary metabolites

Table 2. Investigating chemical compositions of nyamplung kernel cake (%, DM)



Dry matter


Organic matter


Crude protein


Ether extract


Neutral detergent fiber


Acid detergent fiber




DM, dry matter

Concentrations of DM, OM, CP, EE, NDF, ADF, and HEMI from NKC were 90.2%, 92.1%, 20.1%, 15.3%, 53.3%, 38.2%, and 15.1%, respectively (Table 2). The feedstuff from oil extraction commonly contain high concentration of protein. The extraction of oil will decrease the proportion of fat in the kernel and then increase the proportion of protein. In addition, NKC also contained several secondary metabolites such as phenol, flavonoid, total saponin, and tannin (Table 3). Concentrations of total phenol, total flavonoid, and total saponin from NKC are 6.47%, 1.70%, and 0.90%, respectively. The total tannin of NKC is 0.93% consisting of condense tannin at 0.45% and hydrolysable tannin at 0.48%.

Table 3. Secondary metabolites containing in nyamplung kernel cake (%, DM)



Total phenol


Total flavonoid


Total saponin


Total tannin


Condensed tannin


Hydrolysable tannin


DM, dry matter.
Acceptability test

Table 4. Acceptability test of nyamplung kernel cake as animal feed on male sheep (n=30 head)


Consumed NKC

Not Consumed NKC

Day 1st



Day 2nd



Day 3rd



Average, n



Average, %



NKC, nyamplung kernel cake

Total 61.0% of sheep consumed NKC, while Total 39% of sheep did not consumed. This indicated that acceptability of NKC was good enough as a single feed. More than 50% of animals could consumed NKC. And in the field, farmers already apply it as ruminant feed and shows high palatability and acceptability. In day 1st of observation, the NKC was consumed by 18 head of sheep, while in day 2nd was 17 head (Table 4). In the last day of observation, the NKC was consumed by 30 head of sheep.

Ruminal in vitro incubation

Figure 1. Investigating in vitro digestibility of dry matter (A) and organic matter (B) from several feedstuffs. NKC, nyamplung kernel cake;
PKC, palm kernel cake; CC, copra cake; TLM, tamarind leaves meal; GLM, gliricida leaves meal; MKC, malapari kernel cake.
a,b,cMean with different superscripts differ significantly (p<0,05)

In the present study NKC was compared to several feedstuff to evaluate the digestibility in the rumen (Figure 1). In general, the NKC had a similar IVDMD to PKM, CM, and GL. The IVDMD of NKC, PKC, CC, and GLM were 51.2%, 44.4%, 53.5%, and 53.1%, respectively. The IVOMD of NKC was also similar to CC and GLM. The IVOMD of NKC, CC and GLM were 65.9%, 57.2%, and 60.4%, respectively. In addition, NKC had higher IVOMD than PKC (p<0.05; 65.9% vs. 50.2%). The highest of IVDMD and IVOMD was resulted by MKC, while the lowest was resulted by TL (p<0.05). The IVDMD and IVOMD of MKC were 73.0% and 79.1%, respectively. And also, the IVDMD and IVOMD of TLM were 39.9% and 46.3%, respectively.

Figure 2. Methane emission in the rumen from different feedstuffs. NKC, nyamplung kernel cake; PKC, palm kernel cake;
CC, copra meal; TLM, tamarind leaves meal; GLM, gliricida leaves meal; MKC, malapari kernel cake.
a,b,cMean with different superscripts differ significantly (p<0,05)

The NKC had the lowest methane emission compared to all feedstuffs (p<0.05) (Figure 2). On the other side, the methane emission of PKC and CC was lower than TLM, GLM and MKC. During 48 h of ruminal incubation, NKC produce methane at 47810.6 ppm, while the methane emission of PKC, CC, TLM, GLM, and MKC were 55143.7, 55741.3, 771007.8, 69826.1 and 77761.1 ppm, respectively.

Table 5. Fermentation indices in the rumen from different feedstuffs




















Ammonia-N, mg/100 mL









Total VFA, mg/L









Acetate, %









Propionate, %









Iso-butyrate, %









Butyrate, %









Iso-valerate, %









Acetate : Propionate









1NKC, nyamplung kernel cake; PKC, palm kernel cake; CC, copra cake; TLM, tamarind leaves meal; GLM, gliricida leaves meal; MKC, malapari kernel cake. a,b,cMean in the same row with different superscripts differ significantly (p<0,05). SEM, standard error of mean.

In rumen fermentation indices, MKC had the lowest rumen pH, followed by NKC, PKC, and CC, and then by TLM and GLM (p<0.001; 6.68 vs. 7.01, 7.04, and 7.02 vs. 7.26, 7.11) (Table 5). The concentration of ammonia-N by MKC was the highest than other feedstuffs (p<0.001; 16.2 mg/100 mL vs. 7.89, 8.19, 6.63, 9.96, and 9.50 mg/100 mL). Total VFA in the rumen did not affected by feedstuffs. The acetate concentration of NKC was similar to other feedstuffs. The NKC had higher concentration of propionate compared to PKC and CC (p=0.014; 32.9% vs. 28.3% and 28.0%), but similar to TLM, GLM, and MKC. In addition, NKC had lower butyrate concentration than PKC and CC (p<0.001; 25.1% vs. 30.5% and 21.1%), but higher than TLM and GLM (p<0.001; 25.1% vs. 21.1% and 20.4%). In general, the NKC had similar ratio of acetate to propionate compared to all feedstuffs.


The chemical compositions of NKC generally are similar to oilseed by products such as, PKC and CC; and legumes, such as TLM and GLM. the physical appearance of NKC also similar to PKC and CC that have a brown color and dry texture (Figure 1). The NKC contains 20% of CP and 53.3% of NDF. The CP and NDF content of NKC are in the same range concentration with palm kernel meal (Sundu et al 2006), copra meal (Sundu et al., 2009), tamarind leaves (De Angelis et al 2021), and grilicida leaves (Abdulrazak et al 1997). The NKC also had similar characteristics with MKC. The chemical composition of MKC is presented in Table 1. The MKC is also by product of biofuel industry using malapari kernel. The industry of MKC is still developing recently in Indonesia. The main differences among NKC and the other feedstuffs is concentration of EE. The concentration of EE in NKC is higher than in other feedstuffs, which might increase the potential for rancidity (Stein et al, 2005).

Nyamplung kernels are a rich of plant secondary metabolites (Udarno and Tjahjana 2019), which may be remained in their by-product after oil extraction. Thus, the investigation is necessary to confirm it since the plant secondary metabolites can play a role as anti-nutritional factor for ruminant diet (Bodas et al 2012). Previous studies investigated the concentration of plant secondary metabolites in several by-products from oil extraction. The by-products of palm oil such as kernel cake, pressed fibre, and kernel shells have a total phenol lower than 0.50% (Tsouko et al 2019). The concentration of total phenol in rapeseed meal, soybean meal, and sunflower meal is lower than 0.20%. In addition, the tannin and saponin concentrations are less detected in those feedstuffs (Nehmeh et al 2022). According to Archimčde et al (2016), Leucaena leucocephala and Gliricida sepium leaves contains condensed tannins approximately at 8.9% and 1.8% of DM, respectively. Other study reported that total phenol and total tannin in Leucaena leucocephalaleaves are 3.48% and 2.33% of DM, respectively (Soltan et al 2013). This indicated that NKC has higher concentration of plant secondary metabolites compared to other oilseed by-products. However, concentrations of tannin and saponin are still lower than Leucaena leucocephala and Gliricida sepium leaves.

Based on in vitro study, the use of NKC could produce a lower methane emission with similar digestibility and fermentation indices to PKC, CC. and GL. Moreover, the NKC resulted in the lowest methane emission. This indicated that the NKC can replacement or substitution for those feedstuffs for ruminant feed, especially to create a good farming model with low methane emission. High concentration of fibrous plant material can increase the emission of methane in the rumen. While, high concentration of non-fibrous carbohydrates in the diet is reported to decrease methane emission (Jansse 2010; Preston 2023). In addition, the feed containing more protein with less fiber produce lower methane emission. More concentration of fibrous carbohydrate can produce more propionate, which utilize H2and reduce the potency of methane emission (Jansse 2010). This was a cause that NKC had lower methane emission than PCK and CC. In the present study, NKC contained high concentration of fat compared to other feedtsuffs, which could be a reason for the lowest methane emission. Amanullah et al (2022) reported that dietary essential fatty acid could reduce methanogenic bacteria without any effects on fibrinolytic bacteria in the rumen. Another study also reported same result with the present study, which supplementation of fat could result a toxic effects for rumen microbes, such methanogenic bacteria and ciliate protozoa (Vargas et al 2020). By high fat concentration in NKC, the methane production could be decrease without any negative effects on digestibility.

The high concentration of plant secondary metabolites might be also another reason for low methane emission by NKC. Plant secondary metabolites can be act as rumen modifier (Bodas et al, 2012; Paverini et al 2012). Toxic effects of plants secondary metabolites could be varied, which is influenced by the application dose and the types of secondary metabolites (Paverini et al 2012). High dose of plant secondary metabolites causes toxic effect on ruminant. However, by an appropriate dose, dietary plant secondary metabolites can modify the ruminal fermentation to reduce a methane emission in ruminants without any negative effects (Bodas et al 2012).

The use of diet containing plant secondary metabolite could be a strategy to decrease methane emission by ruminants. The NKC contains phenol, flavonoid, tannin, and saponin, which present many beneficial effects to inhibit the growth methanogenic bacteria (Waghorn 2008; Jayanegara et al, 2010; Bodas et al 2012; Lee et al 2021). For the example, dietary olive leaves rich of phenol and flavonoid confirmed to decrease methane emission by in vitro study due to the reduction of methanogenic archaera (Lee et al, 2021). Jayanegara et al (2010) reported that supplementary purified tannin at 1 mg/mL in the diet decreased methane emission through in vitro study. In addition, dietary several saponin-containing plants, such as Sesbania sesban and Trigonella foenumgraecum L. did not effectively reduce a methane emission but could increase the production of microbial mass (Jayanegara et al 2010). According to Bodas et al (2012), saponins have a role as antriprotozal activity and reduce methane emission through in vitro or in vivo study. On the other previous study, the dietary single plants containing high hydrolysable tannins or high condensed tannin reduced the production of methane, but also decreased the fermentation characteristics in the rumen. Combination of plant rich tannin with high quality forage could was reported to reduce a methane emission without any negative effects on rumen fermentation (Jayanegara et al 2013). In addition, condensed tannins could improve animal performance and reduce the negative impact of gastro-intestinal parasitism in appropriate dose through in vivo study (Waghorn 2008). The emission of methane production mainly can be reduced by the presence of tannins in the diet, which in the same time also result in the inhibition of fiber digestion (Vasta et al 2019). Therefore, IVDMD and IVOMD of TLM were the lowest that might be due to a high concentration of tannin in Leucaena leucocephala leaves. However, the hydrolysable tannins potentially decrease production of methane in the rumen without presenting a negative effects of digestibility (Vasta et al 2019). The specific dose for each plant secondary metabolite was unknown yet. The optimum level of application depending on the substrate and the types of secondary metabolites (Bodas et al, 2012; Paverini et al 2012). Reduction of methane emission without any decrease ruminal fermentation is the main goal to create a green farming system. Thus, the NKC can be used as alternative protein source for ruminant that help to reduce methane emission. Application of NKC as single feed in the present study was proven to produce lower methane emission.


The NKC could be used as protein source and have similar chemical compositions to several feedstuffs such as PKC, CC, TLM, GLM, and MKC. In addition, the NKC contained high concentration of fat and plant secondary metabolites, which can help to reduce methane emission. The NKC produced the lowest methane emission compared to PKC, CC, TLM, GLM, and MKC without any negative effects on digestibility. The present study recommend that nyamplung kernel cake can be used as substitution for protein source feedstuffs such as palm kernel cake, copra cake, tamarind leaves, gliricida leaves, and malapari kernel cake.


This study was supported by Program Riset and Inovasi untuk Indonesia Maju/ Research and Innovation Program for Advanced Indonesia (No 82/11.7/HK/2022), National Research and Innovation Agency (BRIN) Indonesia, and Indonesia Endowment Fund for Education (LPDP) Ministry of Finance Republic of Indonesia.

Conflict of Interest

The authors declare no conflicts of interest.


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