Effect of Calliandra calothyrsus on in vitro digestibility of soybean meal and tofu wastes

E Wina, B Tangendjaja and Dumaria

Research Institute for Animal Production, P.O.Box 221, Bogor 16002, Indonesia
winabudi@yahoo.com

Abstract

The present study consisted of two experiments conducted in vitro. The first experiment was aimed to investigate the effectiveness of fresh Calliandra calothyrsus leaves as a tannin source to reduce rumen degradable protein and increase bypass protein of soybean meal and tofu waste (raw and cooked). Calliandra leaves were mixed with tofu waste or soybean meal in the ratio of 0:100, 15:85, 30:70, 45:55 on dry matter  basis and the mixtures were incubated for 48 h in rumen liquor (first stage incubation) followed by further incubation with pepsin-HCl for 24 h (second stage incubation).  The digestibility coefficients of crude protein and ammonia concentration were reduced with increasing levels of calliandra inclusion when mixed with soybean meal and cooked tofu waste. The regression coefficients (r²) of dry matter digestibility coefficients for soybean meal, raw tofu waste, cooked tofu waste at stage 1 and stage 2 of incubations were 0.89, 0.93, 0.98 and 0.98, 0.87, 0.93, respectively, while for the crude protein digestibility coefficients, these were 0.94, 0.95, 0.95 and 0.78, 0.87, 0.83, respectively. The coefficient digestibility of crude protein of the soybean-calliandra (55:45 dry matter) mixture was high (0.89) at the second stage of incubation indicating that there was no inhibition of pepsin digestion due to tannin. 

The second experiment was performed to measure the effect of calliandra leaves in the mixtures with different protein sources (50:50 dry matter) on protein solubility, digestibility, ammonia and tannin content. When calliandra leaves were mixed with tofu waste at 50:50 dry matter, condensed tannin content decreased to around half (39.9 and 46.2 g/kg, respectively for raw tofu waste and cooked tofu waste) and to 21.5 g/kg when mixed soybean meal. The results suggest that there was a higher binding of tannins from Calliandra  leaves with protein of soybean meal than that of tofu waste. There was a strong relationship between total tannin-soluble protein ratio and difference of coefficient of digestibility of crude protein between second and first stage (r²=0.71**), which may be used to predict the amount of bypass protein that can be obtained using tannin. In conclusion, Calliandra calothyrsus can be utilized as a potential tannin source to reduce protein degradability in the rumen and enhance bypass protein of soybean meal and cooked tofu waste. Further in vivo evaluation of these calliandra-protein mixtures is warranted. 

Keywords: bypass protein, ruminal digestibility, soluble protein, tannin

Introduction

Ruminants have the ability to survive on poor quality, roughage-based feed sources because of a symbiotic relationship with the microflora and fauna in the rumen. These rumen microorganisms digest some of the feed and provide the animal with useable metabolic products, especially microbial protein that can be metabolized further down the gut.  However, when fed with high quality diets (25–35 g N/kg dry matter), a large proportion of the protein (56-65%) is rapidly metabolized and released into the rumen resulting in large losses of N as ammonia (25–35%). This imposes an additional cost to the animal due to inefficiencies in microbial protein production (MacRae and Ulyatt 1974, Min et al 2000). Therefore, there is a scope to improve the overall efficiency of animal performance if the readily degradable protein can be protected from degradation in the rumen, but still be digested in the intestine.

The nutritional advantage to animals by manipulation of ruminal protein degradability has been known for many years (Nolan 1975).  The protein in the feed can be protected from rumen degradation, by various methods such as heat, mechanical processes, and chemical treatment such as lignosulfonate and formaldehyde (Broderick et al 1991, Stern et al 1994).  In addition, many plants contain secondary compounds that have the potential to modify protein digestion in the rumen either by direct interaction with the protein or by changing the rumen microbial population. One such group of plant materials that have a known ability to reduce protein degradation is tannins. Both positive and negative effects of tannins on animal nutrition have been reported (Getachew et al 2000, Makkar 2003a, Min et al 2003). In the presence of tannin, the protein may be bound and is protected from microbial degradation but is released and digested in the abomasum and then, the released amino acids are absorbed in the small intestine (Barry and Manley 1984, Barry and McNabb 1999). However, the proteins may be overprotected and excreted in the faeces resulting in poor animal production (Getachew et al 2000, Makkar 2003a). Calliandra calothyrsus is a widespread leguminous shrub in many parts of Asia. Although high in protein, it also contains high concentration of condensed tannins (Palmer et al 2000, Lascano et al 2003). However, a potential use of tannin in Calliandra calothyrsus in relation to its property to bind with other protein sources to increase rumen undegradable protein was limitedly reported. An isolated tannin of Calliandra calothyrsus had the same ability as formaldehyde to bind forage protein (Gliricidia sepium) (Wina and Abdurohman 2005)

Tofu waste, like soybean meal, is a good protein source, which is readily available in Asia. Tofu is made from soybean by extraction and precipitation to produce a semi-solid product rich in protein. During the tofu preparation, the extracted protein, may or may not be heated, and is separated by filtration. The residues (waste products), with high protein and fiber contents, are called raw  or cooked tofu waste  from the unheated and heated treatments, respectively. In some countries, it is called okara and has a protein content of approximately 250 g/kg (van der Reit et al 1989). Utilization of soybean meal as feed for ruminant is a common practice and there are reports suggesting methods to reduce rumen degradability of soybean meal protein (Spears et al 1985, Ivan et al 1996, Hervas et al 2000). Although in some parts of Asia, tofu waste is used as animal feed (Amaha et al 1996), there is only limited information on rumen degradability of tofu waste protein.

This study was carried out to assess the potential use of Calliandra calothyrsus, as a tannin source, for improving the digestive utilization of tofu waste and soybean meal.

Materials and methods  

The present study consisted of two in vitro experiments, which conducted three times with two replicates for each treatment. In the first experiment, the effect of different levels of calliandra and different protein sources (0:100, 15:85, 30:70, 45:55 on dry matter basis) on coefficient of digestibility of dry matter and crude protein and ammonia concentration were measured. The second experiment was performed to measure the effect of 50:50 dry matter calliandra leaves in the mixtures with different protein sources on protein solubility, digestibility, ammonia and tannin content.

Plant and protein sources

Leaf samples were obtained from Calliandra calothyrsus shrubs, growing 0.5 m apart in rows separated by 1 m at the Research Institute for Animal Production, Ciawi - Bogor, Indonesia. Samples were taken immediately to the on-site laboratory where they were chopped and used directly or mixed with the appropriate proportions of other protein sources for the experiments. The protein materials used in the experiments were all derived from soybeans. Dehulled soybean meal from South America was milled in Indonesia, and two other protein materials, raw tofu waste and cooked tofu waste, were obtained in a wet state from a local tofu manufacturer.  These protein-containing materials were transported to the laboratory within one hour and were stored in a freezer at –20^oC until required.

Preparation of the mixtures for in vitro studies

Various amounts of fresh chopped calliandra leaves were mixed in a blender for 5 minutes with either cooked tofu waste or raw tofu waste or soybean meal in ratio of 0:100, 15:85, 30:70 and 45:55 on dry matter basis.  The previously determined dry matter content of the different materials (Table 1) was used to calculate the required weights of the fresh samples. 

For example: to prepare 100 g dry matter of a mixture of calliandra: cooked tofu waste (15:85 dry matter), the amount of fresh calliandra leaves (dry matter 374 g/kg) required = 15 * 100/37.4 = 40.1 g and the amount of cooked tofu waste (dry matter 101 g/kg) required = 85 * 100/10.1 = 841.6 g. Weights of calliandra leaves and other protein sources for other mixtures were calculated in the same way.

Sub samples of the original materials and the fresh mixtures were used in the in vitro incubation studies.

Digestibility

In vitro digestibility was determined using a modified Tilley and Terry (1963) two-stage digestion method in which Stage 1 was 48 h incubation using rumen liquor and Stage 2 was stage 1 followed by 24 h incubation using pepsin in acidic solution.  Dry matter and nitrogen (N) were determined in the original materials and in the residues after each stage, and the values were used to calculate coefficient of digestibility of dry matter and crude protein.

Protein and ammonia analysis

Nitrogen in the samples was determined using the Kjeldahl method.  Digestion by sulfuric acid was carried out as described in AOAC (1984), and ammonia level was determined using the Conway method (Conway and Byrne 1933).  Soluble protein in the samples was determined after extraction of samples (1 g dry matter) in 40 ml artificial saliva buffer using a blender for 5 minutes and then centrifuged at 3000 g for 10 minutes. The soluble protein content in the supernatant (5 ml) was determined using the Kjeldahl method.

Tannin analysis

Total tannins  and condensed tannins  were determined respectively, using modified Folin Ciocalteau and butanol-HCl methods as described by Makkar (2003b).

Statistical analysis

Results from triplicates of in vitro incubation were analyzed using proc General Linear Model (GLM) SAS program version 6. The effect of levels of calliandra in the mixtures was examined by standard linear regression analysis and the Duncan multiple range test was used to compare treatment means.

Results  

Composition of materials

Dry matter and protein contents of Calliandra calothyrsus, raw and cooked tofu waste and soybean meal are presented in Table 1.  Since tofu waste was obtained directly from tofu manufacturer without drying, it had high moisture content. The dehulled soybean meal was in an air-dried form. The protein level of soybean meal was more than double that of the other protein sources (Table 1).

Effect of different levels of calliandra leaves on digestibility coefficient of three protein sources

Digestibility coefficient of dry matter of the different mixtures of protein sources with calliandra is presented in Figure 1.

The raw tofu waste had the highest digestibility coefficient of dry matter (0.88) followed by cooked tofu waste (0.81) while soybean meal has intermediate coefficient of digestibility at 0.85 at the first stage of incubation. Increasing levels of calliandra leaves resulted in a linear decrease of digestibility coefficient of dry matter in both first and second stages of the Tilley and Terry technique. 

The rates of decline were similar for the different protein sources in the 48 h of incubation. Digestibility coefficient of dry matter declined by approximately 0.005 for every portion of calliandra added into the mixture. However, after addition of pepsin for the second stage incubation, the rate of coefficient digestibility of dry matter for soybean meal mixture declined by 0.004, while that for the tofu waste materials decreased by 0.006 for every addition of calliandra.

The coefficient digestibility of crude protein of the mixtures also declined linearly with increasing levels of calliandra in both stages (Figure 2). 

However, in the first stage, the rate of decrease in coefficient digestibility of crude protein was the same to that of dry matter for raw tofu waste (0.005), but was higher for cooked tofu waste (0.01 vs 0.006) and soybean meal (0.009 vs 0.006).  In the second stage, only the rate of coefficient digestibility of crude protein for soybean meal was slower than that of dry matter (0.003 vs 0.004).

Ammonia was produced as the end products of protein degradation or microbial lysis. Table 2 shows that the in vitro ammonia concentration declined as the proportion of calliandra in all diets increased, from 38.8 mM in 0 % calliandra to 15.6 mM in 45% calliandra for the first stage of incubation.

A dramatic drop of ammonia concentration was found in 45% calliandra. In both stages, the ammonia from soybean meal showed a relatively steady decline to about 50% of its initial value, but the ammonia concentration with cooked tofu waste and raw tofu waste were more variable, with a much greater decline and a dramatic drop at a calliandra :protein level of 45:55. Different protein materials had a significant effect to ammonia concentration.  Ammonia concentration for soybean meal was much higher than that for cooked tofu waste or raw tofu waste, while ammonia concentration for raw tofu waste was higher than that for cooked tofu waste.

Effect of a mixture of calliandra leaves with different protein sources (50:50 dry matter) on coefficient digestibility

The proportions of the total protein that are soluble in buffer solution, in the different mixtures showed that calliandra leaves had relatively low soluble protein level (43.8 g/kg) and are similar to that in soybean meal. But cooked tofu waste and raw tofu waste had soluble protein levels about 1.5 and 4 fold higher soluble proteins than soybean meal (Table 3).

Total tannin  and condensed tannin  contents in Calliandra were 130 and 68 g/kg, while the corresponding tannins in the soy-based materials were negligible (Table 3). When calliandra leaves were mixed with tofu waste at 50:50 (calculated on dry matter basis), the total tannin content decreased to around half of the original values as expected due to dilution.  However, when mixed with soybean meal, the tannin content in the mixture was only half of that in the mixture of calliandra-tofu waste.

Fresh calliandra leaves had a low digestibility coefficient of dry matter (0.36) following rumen fluid incubation, which increased to 0.50 after pepsin-HCl incubation (Table 4).

In contrast to calliandra, the other protein sources, tofu and soybean meal, had very high digestibility coefficient of dry matter 0.94 (first stage), and there was no significant increase in coefficient of digestibility of dry matter after pepsin digestion (second stage). The mixture of calliandra and protein sources resulted in a decrease in digestibility coefficient of dry matter of the mixtures and the observed values, especially those for the tofu wastes, were very close to the values calculated using the relative contributions from each source. The difference of coefficient digestibility of dry matter between first and second stage of incubation of the mixture of calliandra with soybean meal was the highest (0.18) compared to those of the mixtures with tofu waste (0.09 and 0.08 for raw tofu waste and cooked tofu waste, respectively).

The concentration of ammonia  from the calliandra/soy-protein mixtures declined by more than 50% compared with the original soy-protein over 48 h incubation (Table 5). 

Calliandra protein was poorly digested in the first stage of incubation (0.03) but the coefficient value increased significantly at the second stage of incubation (0.46) (Table 5).  The other protein sources on their own were completely digested by pepsin. The digestibility coefficient of crude protein at the first stage of incubation declined when the protein sources were mixed with calliandra, the lowest value was for the Calliandra + cooked tofu waste (0.31), intermediate for Calliandra + raw tofu waste (0.46) and the highest Calliandra + soybean meal (0.50).  The difference in the values for coefficient of digestibility of protein between first and second stage of incubation was about 0.30 for all the calliandra and protein source mixtures.

A quadratic response (r² = 0.71, P < 0.001) was found between the difference of  digestibility coefficient of crude protein of second and first stage of in vitro rumen fermentation and the ratio of total tannin to soluble protein. However, this quadratic response had not reached the maximum at a ratio of 1.53 (Figure 3).

When total tannin was replaced by condensed tannin, a similar correlation strength was found (r² = 0.71). When the correlation was calculated between the difference between digestibility coefficient of crude protein of second and first stage of in vitro rumen fermentation and the ratio of total tannin to crude protein, instead of the total tannin to soluble protein ratio, a poor quadratic relationship was found (r² = 0.29).

Discussion 

Increasing the calliandra level with all protein sources decreased the digestibility coefficient of dry matter and  CP both after 48 h (1^st stage) and 72 h of incubation (Figure 1 and 2). During the mixing process of calliandra leaves with the protein sources, the plant cells rupture and release tannins from the vacuole (Getachew 1999). These tannins may quickly bind to the protein from calliandra leaves and also to the protein from soybean meal or tofu waste particularly at higher levels of inclusion of calliandra. Barry and McNabb (1999) reported that tannins preferably react with proteins in the forage of the tannin-containing plant than with proteins from associated non tannin-containing plant materials. Beneficial effects of forage mixing are expected if the tannin content is extremely high, thus releasing some ‘free’ tannins to bind with proteins from other sources. The effect of tannin from calliandra was more marked for protein than for dry matter since there was a strong hydrogen bond affinity of the phenolic groups of tannin molecule with the carbonyl oxygen of the peptide group (McLeod 1974). With the increasing level of calliandra, a lower digestibility coefficient of crude protein occurred mainly due to a lower rate of degradation and only partially due to a reduction of rapidly degradable fraction (Frutos et al 2000). Consequently, the reduction of crude protein digestibility in the rumen resulted in a reduced ammonia level in a dose dependant manner (Table 2).

Several results on the reduction of ruminal nitrogen degradability of soybean meal using tannin have been reported (Driedger and Hatfield 1972, Frutos et al 2000, Hervas et al 2000). Nitrogen degradability of soybean meal was reduced when quebracho tannin was added at the level above 150 g/kg dry matter (Frutos et al 2000) while Driedger and Hatfield (1972) reported that the least degradable soybean meal protein occurred when tannic acid was added at the level of 100 g/kg.  Hervas et al (2000) found a dose dependant effect of tannic acid on the reduction of ruminal N degradability of soybean meal. Different results on the effect of tannin on N degradability might be due to several factors such as the concentration of tannin (Driedger and Hatfield 1972, Baharona et al 1997, Frutos et al 2000), the chemical structure of tannin (Aerts et al 1999; Lascano et al 2003) and the type of protein (Hagerman and Butler 1981, Asquith and Butler 1986, Poncet and Remond 2002).

A larger magnitude of difference in coefficient digestibility of crude protein between stage 1 and stage 2 of incubation at 45:55 dry matter of calliandra leaves in the mixtures with soybean meal and cooked tofu waste indicates more complex between calliandra tannins to the protein source during mixing. The second experiment with 50:50 of calliandra : other protein source confirmed that there was a higher binding between calliandra tannins with soybean meal than with tofu waste and there is a little difference between cooked and raw tofu waste.  Although calliandra was only 50 % in the mixture (dry matter basis), there was a reduction of more than 50% in the tannin content after mixing with soybean meal (Table 3). This result shows that more tannins in calliandra bind with protein in soybean meal and more protein might be released at low pH from their complexes with tannins and digested by pepsin. Tannins in calliandra did not suppress the intestinal digestion. The same result has been reported earlier that low doses of tannic acid or quebracho tannin did not negatively affect the intestinal digestion of soybean meal in sheep (Frutos et al 2000, Hervas et al 2000).

If the rumen undegradable protein is defined as the difference between protein digested in rumen fluid at 48 h (first stage) and protein digested by pepsin (second stage), the results suggest that the addition of calliandra especially in a 45:55 or 50:50 ratio in the mixture produced higher rumen undegradable protein from soybean meal, which is beneficial for ruminant.

The observed differences between soybean meal: calliandra and tofu waste : calliandra may be due to the different chemical composition and characteristics between tofu waste and soybean meal. Soybean meal was a dehulled solvent extracted soybean, therefore, had very high protein content (>500g/kg) and less NDF content (100 g/kg). There was no loss of soluble and fermentable components from soybean meal as the fat extraction was done by the non-polar organic solvent. Tofu waste, on the other hand, still contained all the soybean hulls (high fibre) with very low content of soluble and fermentable components. Tannin may get bound to the fibre fractions and protein during a longer incubation and could affect the pepsin digestion of tofu waste. The presence of tannin-protein complexes in fibre fractions has been reported previously (Makkar et al 1995) and these complexes may not get dissociated in the post rumen.

From this experiment, we found a strong relationship (Figure 3) between ratio of total tannin /soluble protein and the rumen undegradable protein (difference between first and second stage of incubation).  With the quadratic relationship, we could calculate the ratio of total tannin / soluble protein for obtaining a maximum rumen bypass protein (0.40) and that was 1.9. It is also important to consider not only the amount of tannin but also the amount of soluble protein in the diet. However, when the ratio of tannin to soluble protein becomes higher (> 1.9), the amount of bypass protein would decrease. When the ratio of tannin to soluble protein is higher than 1.9, it suggests that there are more tannins  present in the rumen, which may interfere the attachment of microbes to feed particle by complex with microbial enzyme (McAllister et al 1994). Tannins may also flow out from the rumen to the intestine and may induce a depressive effect on the intestinal enzymes activity of trypsin and amylase (Silanikove et al 1994).  

Conclusions 

• From this experiment, it is suggested that Calliandra calothyrsus can be used to reduce protein degradability in the rumen and enhance availability of protein postruminally from soybean meal and to less extent from cooked tofu waste. 

  
  

• In vivo evaluation using mixtures of calliandra with protein sources is warranted to improve animal performance.

Acknowledgement 

We thank the International Atomic Energy Agency (IAEA) for the financial support for the studies and Ms. Dumaria for assistance in the analyses.

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Received 7 February 2008; Accepted 4 March 2008; Published 10 June 2008

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