Effects of fungal treatment on the in vitro digestion of palm kernel cake

M Ramin, A R Alimon^* and M Ivan^*,**

Department of Animal Science, Faculty of Agriculture, University of Putra Malaysia,
43400 UPM Serdang, Selangor, Malaysia
mramin1981@gmail.com
* Institute of Tropical Agriculture, University of Putra Malaysia,
43400 UPM Serdang, Selangor, Malaysia
** Dairy and Swine Research and Development Centre, Agriculture and Agri-Food Canada, 2000 College Street, PO Box 90 STN Lennoxville, Sherbrooke, Quebec, Canada J1M 1Z3

Abstract

Palm kernel cake (PKC) was fermented for 10 days under solid state fermentation culture with three different fungi (Aspergillus niger, Trichoderma harizianum or Rhizopus oryzae) to increase its nutritional value.

There was increase (P < 0.05) in crude protein concentration and decrease (P < 0.05) in both neutral detergent fiber and acid detergent fiber concentrations due to treatment of PKC with Aspergillus niger or Rhizopus oryzae. The treatment with  Trichoderma harizianum  was not effective and, therefore, was not used in a follow-up in-vitro gas production experiment. The gas production was greater (P < 0.05) from the fresh PKC compared to PKC fermented for 10 days with Aspergillus niger or Rhizopus oryzae.

It was concluded that Aspergillus niger or Rhizopus oryzae are potentially effective fungi for treatment to increase the nutritional value of PKC.

Keywords: Gas production, PKC, Solid state fermentation

Introduction

Palm kernel cake has the highest nutritive value among the palm oil byproducts and is widely used in diets of ruminants (Babjee 1989). The crude protein (CP) content of PKC varies between 10 and 16% (Yeong and Mukherjee 1983). However, the components of carbohydrate in PKC include cellulose, β-mannans and lignin. These components result in low metabolisable energy of PKC. There are many treatments available to break down the cellulose chain in PKC to make it more digestible. The chemical and physical methods do not appear to improve the nutritional value of PKC. However the solid state fermentation (SSF) and enzyme treatments are potentially beneficial. A number of studies have shown that using enzyme producing microbes in SSF increases the protein value and the bioavailability of nutrients (Fernandez et al 1989), but the most suitable microorganisms for treatment of PKC have not been identified. Treatments with microbes such as fungus and bacteria have shown positive effects on cellulose digestion (Ramin et al 2008). Consequently, it was the objective of the present experiment to identify a fungus with the potential to improve the digestibility and protein value of dietary PKC. Three fungi species (Aspergillus niger, Trichoderma harizianum and Rhizopus oryzae) were used for this purpose. Because in vivo experiments might not be the most suitable for evaluation of the feed treatment benefits (Gooselink et al 2004), an in-vitro gas production technique was utilized in the present experiment. Such technique has the ability to characterize feeds not only by the quantity of digestible carbohydrates they provide, but also by the rate at which the carbohydrates are released (Nagadi et al 2000).

Materials and methods 

Solid state fermentation

Three species of fungi (Aspergillus niger, Trichoderma harizianum and Rhizopus oryzae) were obtained from the University Department of Plant Protection, sub cultured on the potato dextrose agar and kept refrigerated at 4°C. Palm kernel cake was obtained form the Department of Animal Science Farm, grinded to pass 1mm sieve and oven dried at 60ºC. The nutrition component of fresh PKC before fermentation was 13% CP, 74% NDF and 42% ADF. Dry PKC (100 g) was placed into each of thirty six 500 ml Erlenmeyer flasks, followed by 9 g of (NH₄)₂SO₄, 2.7 g of urea, 5 g of KH₂PO₄ and 50 ml of distilled water. The flasks were covered with cotton plugs and sterilized at 120°C for 20 min. They were then divided into four groups of nine flasks each and each group was assigned one of four treatments 1. PKC without fungus (control (CN)), 2. PKC + Aspergillus niger (AN) 3. PKC + Trichoderma harizianum (TH) and 4. PKC + Rhizopus oryzae (RO). Approximately 1 cm² of corresponding fungus was inoculated into each of nine flasks in treatments 2, 3 and 4 to prepare fresh SSF cultures. Three cultures (replicates) in each treatment were fermented for each 4, 7 and 10 days at 35ºC. After completion of fermentations the content of each flask was removed and oven dried at 60ºC for 48 h. The contents were then grinded using a blender, homogenized, and stored at 4ºC for further analysis. The CP concentration in the contents was determined as Kjeldahl N × 6.25. The acid detergent fiber (ADF) and neutral detergent fiber (NDF) were analyzed as described by Van Soest (1963).

In-vitro gas production

Fresh PKC (control) and PKC after 10 days fermentation with Aspergillus niger and Rhizopus oryzae from the previous experiment were used as substrates in the current in-vitro gas production experiment. 10 day fermentations were selected because on completion they produced higher CP and lower ADF and NDF concentrations than the other fermentations. The dry matter (DM) loss, concentration of volatile fatty acids (VFA) and cumulative gas production in the current in-vitro gas production experiment were measured according to Menke and Steingass (1988). Rumen contents were collected in pre-warmed flasks 2 h before morning feeding from three fistulated goats fed a normal diet (60% hay and 40% concentrate). Rumen fluid was obtained by squeezing rumen contents through four layers of cheesecloth. The strained fluid was mixed (1:3 v/v) with anaerobic medium to form suspension as described by Menke and Steingass (1988). The suspension was then heated and kept at 39°C for approximately 20 min under continuous bubbling of CO₂. Each appropriate substrate (500 mg) was weighed into 100 ml syringe (Fortuna, Hiberle Labortechnik, Germany) and 40 ml of the suspension was added to each of the syringes. The syringes were incubated for 24 h at 39°C. The cumulative gas production was recorded in each syringe at 2, 4, 8, 12 and 24 h of incubation. The content of each syringe was then transferred to a glass bottle and after the measurement of pH it was used for analysis of VFA as described by Cottyn and Bouque (1968). The molar concentration of acetic acid, propionic acid and butyric acid were determined by a gas chromatograph (Agilent 6890 N) fitted with a flame ionization detector (FID). The length of the column (DB-FFAP, 122-3232) was 30 m and the film was 0.25 µm. The oven temperature was 90-180ºC, nitrogen was a carrier gas, and the FID temperature was 250ºC.

Kinetics of gas production

The rate of gas production was calculated by using NOWAY program (Rowett Research Institute) as described by Saez et al (2005) with the model

Y=a+b (1-exp ^-ct)

Where:

Y is gas volume at time t,
a+b is potential gas production (ml/g DM) and
c (h^-1) is the rate at which gas is produced (Kiran and Krishnamoorthy 2007).

Statistical analysis

The ANOVA was performed on all data using a statistical package of SAS (1997). The means were compared by Duncan multiple range test and considered significant at P < 0.05.

Results 

Solid state fermentation

There was no effect (P < 0.05) of the length of fermentation (4, 7 or 10 days) on the concentrations of CP, NDF and ADF in PKC of the CN treatment (Table 1).

It was though apparent that the CP concentration means in all treatments increased somewhat with increased fermentation time, but the differences were significant only between the fermentation day 4 and other fermentation days (7 and 10) for the AN and the RO treatment, but the differences between the fermentation day 7 and day 10 were not significant. The TH treatment showed higher (P < 0.05) CP concentration after the 10 day fermentation (22.8%) than after the 4 or 7 day fermentation (18.4% and 19.6%, respectively), while the differences were not significant between the 4 and 7 day fermentation. At the 10 day fermentation higher (P < 0.05) and almost identical CP concentrations were obtained for the AN and the RO treatments (27.2% and 27.1%, respectively) than for the TH (22.8%) or the CN (18.7%) treatment. Significant differences in CP concentration between the CN treatment and the AN and the RO treatments persisted throughout the experiment (Figure 1).

Figure 1.  Effects of fungal cultivation (Aspergillus niger, Rhizopus oryzae or Trichoderma harizianum)  under solid state condition on the concentration of crude protein (A), neutral detergent fibre (B) and acid detergent fibre (C) in palm kernel cake at 4, 7 and 10 days of fermentation

The concentrations of NDF and ADF for the CN treatment at all fermentation days were similar (P > 0.05) to those for the TH treatment (Table 1). Similarly, there were no significant differences between the AN and the RO treatments. The concentrations of NDF at all days of fermentation were higher (P < 0.05) for the TH treatment than for the other fungi treatments (AN and RO). The lowest NDF concentration (54.5%) was achieved with the RO treatment at 7 days fermentation and the highest (76.4%) for the TH treatment at 4 days fermentation. The ADF concentration ranged between 36.0% (AN at 4 day fermentation) and 46.3% (TH at 4 day fermentation). There was no particular pattern (Figure 1) of significant treatment effects on the ADF concentration in PKC (see Table 1).

In-vitro gas production

There were no differences (P > 0.05) in pH among the treatments (Table 2).

The total gas production and percentage DM loss were highest (P > 0.05) for the fresh PKC (control) and lowest (P > 0.05) for the Aspergillus niger – treated PKC, but the percentage DM loss for the control was similar (P > 0.05) to that for the Rhizopus oryzae -treated PKC. The concentrations of VFA (acetic, propionic, butyric) were higher (P < 0.05) for the fresh PKC than for the treated PKC with Aspergillus niger or Rhizopus oryzae, while the differences between the two fungi treatments of PKC were not significant.

The gas production during incubation is shown in Figure 2.

Figure 2.  The gas production from fresh (A) palm kernel cake (PKC) and from PKC originating from a 10-day fungal cultivation with Aspergillus niger (B) or Rhizopus oryzae (C)

The rate of gas production (Table 3) was highest (P > 0.05) for the fresh PKC and lowest (P > 0.05) for the Rhizopus oryzae – treated PKC, while that for the Aspergillus niger – treated PKC was intermediate (P > 0.05).

Metabolisable energy and organic matter digestibility is also given in table 3.

Discussion   

The present results showed that from the three species of fungi (Aspergillus niger, Trichoderma harizianum and Rhizopus oryzae) used to treat PKC only two (Aspergillus niger and Rhizopus oryzae) clearly showed better results (increased concentrations of CP and decreased concentrations of ADF and NDF) than when untreated fresh PKC was used under the SSF. The results for the Trichoderma harizianum – treated PKC were similar to the untreated fresh PKC. It was for this reason that both Aspergillus niger and Rhizopus oryzae– treated PKC were selected for further in vitro fermentation study.

After 10 days of SSF both Aspergillus niger and Rhizopus oryzae treatments increased the CP concentration in PKC from approximately 18% to 27% and decreased the concentrations of NDF and ADF from approximately 74% and 43% to 56 % and 37%, respectively. Khin (2004) reported the same increase in CP when Aspergillus niger was used for the fermentation of PKC, while Iluyemi et al (2006) reported a greater increase (33%) when PKC was cultured under SSF.

The amount of gas released when PKC was fermented for 10 days with Aspergillus niger and Rhizopus oryzae was lower than that for control fresh PKC. This is probably due to production of statins by the fungi during fermentation of PKC. Wolin and Miller (2006) reported inhibition of growth of methanogens in the rumen when hydroxymethylglutaryl-SCoA (statins) was used as an inhibitor, while there was no effect on growth of cellulolytic bacteria. This is consistent with the present results showing lower gas production from the fermented than from the fresh PKC. It can be concluded from the results of the present experiment that Aspergillus niger and Rhizopus oryzae are potentially effective fungi for treatment to increase the nutritional value of PKC. Additional in vivo experiments will be conducted in this laboratory to further evaluate these fungi for treatment of PKC as the dietary ingredient for ruminants.

Acknowledgments 

The authors wish to thank Mr. Saparin and Mr. Zakaria for their technical assistance and the Academic Science Grant Scheme for financial support.

References 

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Received 10 February 2010; Accepted 13 March 2010; Published 1 April 2010

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