Effect of Solid State Fermentation on Nutrient Content and Ileal Amino Acids Digestibility of Canola Meal in Broiler Chickens

Abstract

The aim of the current study was to investigate the potential of Lactobacillus salivarius solid state fermentation for reduction of glucosinolate content in canola meal (CM) as well as the improvement of its nutrient digestibility for broiler chickens. Canola meal was treated with the L. salivarius in solid state fermentation for 30 days. Nutrients ileal digestibility was tested using 42-day-old broilers fed by either CM or fermented CM (FCM) as the sole source of energy and protein. The results showed that fermentation of CM using L. salivarius reduced glucosinolate content of CM by 38%. The digestibility coefficient was improved significantly for crude protein, Met, Cys, Arg, Asp, Glu, and Ser in FCM compared to CM. However, apparent metabolisable energy of CM was not affected by fermentation. It appears that fermentation treatment of CM using L. salivarius may improve the overall nutritive value of CM for broiler chickens, reducing its total glucosinolate and crude fibre content by 38 and 16%, respectively.

Key Words:

• Fermentation
• Canola meal
• Broiler
• Digestibility

Introduction

Canola is a particular type of genetically altered rapeseed with <2% erucic acid and <30 µm/g glucosinolate in air-dried oil-free meal (Bell, 1993). Canola meal (CM) is a by-product of canola seed-crushing after oil extraction process and contains high protein (40%) with a well-balanced amino acid (AA) composition (Newkirk, 2009). However, the high fibre and glucosinolate content limits the use of CM in chicken diets. The indigestible carbohydrate content of CM is high (Kocher et al., 2000). Bell (1993) reported that CM has an average 2.5% α-galacto-oligosaccharides and 18% nonstarch polysaccharides (NSP) of which 1.5% is soluble. The soluble NSP tends to increase the digesta viscosity and reduce nitrogen digestion and absorption (Annison, 1991), subsequently resulting in poor growth performance. Moreover, glucosinolates in CM also reduce growth performance by decreasing voluntary feed intake, interfering with the synthesis of thyroid hormones, damaging liver and kidney, and causing anemia (Tripathi and Mishra, 2007). Inclusion of CM at 20% in diets of poultry has been reported to reduce apparent metabolisable energy (AME) (Mushtaq et al., 2007) as well as nutrients digestibility (Slominski and Campbell, 1990).

Solid state fermentation is regarded as one of the possible approaches to diminish the antinutritional factors and enhance the bioavailability of nutrients. Lactic acid fermentation may modify cereal carbohydrate composition as a result of microbial metabolism (Marklinder et al., 1996; Al-Asheh and Duvnjak, 1995). Skrede et al. (2003) indicated that fermentation decreased crude fibre (CF) content of wheat and barley and increased total carbohydrates and starch digestibility. Pal and Walia (2001) reported that solid state fermentation of rapeseed meal using Rhizopus oligosporus reduced its glucosinolate content by 45%. This reduction may occur due to utilisation of glucose and sulphur moieties of these compounds by the fungus. Fermentation by lactic acid bacteria (LAB) also reported to reduce cyano toxicants in jojoba seed meal and trypsin inhibitor in black bean and soybean meal (Granito et al., 2002; Gao et al., 2013). A pilot study in our laboratory (unpublished) showed that fermentation of CM by Lactobacillus salivarius reduced CF and glucosinolate content. Yang et al. (2006) also indicated that fermentation of food waste using L. salivarius enhance breakdown of fibre. However, to the best of our knowledge, the effect of L. salivarius fermentation on nutrient digestibility of CM in broiler chickens is lacking. Hence, the present study investigated the potential of L. salivarius solid state fermentation for reduction of glucosinolate content in CM as well as improvement of its nutrient digestibility for broiler chickens.

Materials and methods

Fermentation procedure

Twenty kg of CM (Brassica napus) were fermented for 30 days using L. salivarius. The L. salivarius was an isolate from Malaysian fermented soybean (tempeh) (GenBank accession number: KF303794). The meal was inoculated with freeze dried L. salivarius at 0.1% based on dry matter (DM) content. The inoculation was first prepared by suspending the appropriate weight of L. salivarius powder in water to increase the moisture content of CM to 70% (Rodriguez-Leon et al., 2008). The inoculant suspension was sprayed over the CM and mixed thoroughly. The colony forming units (cfu) of L. salivarius was 10⁷/g CM. The treated CM was placed in plastic barrel, sealed and incubated for 30 days at room temperature (28 to 32°C). Control CM was prepared at the same time without inoculant addition. At the end of the fermentation period, samples of the untreated and inoculated CM were dried at 50°C for 3 days and ground for chemical analyses. The pH value and cfu were determined at the initial and final stage of fermentation using a portable pH meter (Hanna Instruments, Woonsocket, RI, USA).

Birds and experimental procedure

All experimental procedures were conducted in accordance with Universiti Putra Malaysia Research Policy on animal care. A total of 50 day-old male broiler chickens (Cobb 500) were obtained from a local hatchery and raised in groups of 5 in 10 battery cages with wire floors in a conventional open-sided house (maximum 34°C and minimum 24°C). Chicks were fed commercial broiler starter in crumble form [2900 kcal ME/kg; 21% crude protein (CP)] and grower in pellet form (3050 kcal ME/kg; 19% CP) from day 1 to 21, and day 22 onwards, respectively. Water was available at all times. On day 42, 36 chickens were chosen for digestibility study according to their body weight (2200±50 g). This selection was to eliminate misleading factor of body weight. Groups of three birds were then assigned randomly to 12 cages. All birds were allowed a 4-day adaptation period where they were fed a corn-soybean based diet with 30% CM (basal diet, Table 1) (Soleimani et al., 2010). Two experimental diets containing either CM or fermented CM (FCM) as the sole source of energy and protein were prepared (Table 1) (Ahmed et al., 2014; Jia et al., 2012). Both diets contained titanium dioxide (0.5%) as an indigestible marker. Following the adaptation period, the birds were fasted for 24 h and then allowed to consume the experimental diets (either CM or FCM base, Table 1). Birds were slaughtered 4 h after the beginning of feeding by halal neck cut for collection of ileal content. The 4 h sampling time has been shown to be optimal for ileal digesta sampling in broiler chickens (Kadim and Moughan, 1997). Upon complete immobilisation, the body cavity was opened, and the ileum (from Meckel’s diverticulum to a point 40 mm proximal to the ileocaecal junction) was removed. The ileum was then divided into two parts, and the contents of the lower half of the ileum were collected by gentle flushing with distilled water from a syringe into a plastic container. Digesta samples of birds within a cage were pooled, freeze-dried and stored at -20°C.

Sample analyses

The samples were finely ground using a coffee grinder (Panasonic, Osaka, Japan) and DM, CP, ether extract (EE), CF and ash were determined according to AOAC methods 925.09, 988.05, 920.39, 978.10 and 942.05, respectively (AOAC, 1990). Gross energy (GE) was measured using an adiabatic oxygen bomb calorimeter (C 2000 basic; IKA, Staufen, Germany). Total glucosinolate was determined based on alkaline degradation and subsequent reaction of released 1-thioglucose with ferricyanide (Jezek et al., 1999; Gallaher et al., 2012). Amino acid concentrations in the diet and ileal digesta were determined by high performance liquid chromatography according to the procedures described by Strydom and Cohen (1994). Pre-column derivatisation was done with ACCQ reagent (6-aminoquinolyl-Nhydroxysuc-cinimidyl carbamate; Waters Corporation, Milford, MA, USA). Cys and Met were analysed as cysteic acid and methionine sulfone by oxidation with performic acid for 16 h at 0°C and neutralisation with hydrobromic acid before hydrolysis. Trp contents were determined following alkaline hydrolysis of the sample with 4.3 M LiOH.H₂O for 16 h at 120°C and neutralisation with 6 M HCl. Quantification of the other AA was done by hydrolysing the sample in 5 mL 6 M HCl for 22 h at 110°C. Titanium dioxide was determined according to procedures described by Short et al. (1996).

Calculations

The AME and nutrient digestibility of diets were determined using the following formulas on DM basis:

AME (kcal/kg diet)=GE_diet– [GE_digesta×(marker_diet/marker_digesta)] (Scott et al., 1998);

Digestibility(%)=100-[(marker_diet/marker_digesta)×(nutrient_digesta/nutrient_diet)×10] (Driver et al., 2006);

The AA output and digestibility were calculated using the following equations:

AA output (mg/kg DM intake)=AA_digesta×(marker_diet/marker_digesta);

Apparent AA digestibility (%)=AA_diet–(AA output/AA_diet×100) (Kadim and Moughan, 1997)

Statistical analysis

All statistical analyses were carried out with the Student’s t-test using TTEST procedure of SAS (SAS, 2002).

Results and discussion

The pH values determined at the initial and final stage of fermentation were 5.6 and 4.0, respectively, and cfu were 2×10⁹ and 1×10⁵, respectively. The final pH was within the range of LAB fermentation in silage making (Filya et al., 2007), and the reduction in cfu was as expected due to the diminishing nutrients for bacterial growth. Our results also showed that CM fermentation by L. salivarius increased CP and decreased CF and glucosinolate (P<0.05) (Table 2). However, there were no significant differences between AA content, ash, EE and GE of CM and FCM. Enhancement of CP in FCM in the present study is in agreement with report of Rozan et al. (1996) and it may explained by the loss of DM at the expense of fermentable sugars during fermentation process with LAB. The positive effects of L. salivarius in CF reduction may be attributed to the presence of polysaccharidases as studies of Skrede et al. (2001, 2003) confirmed that fermentation by LAB successfully reduced levels of total and soluble dietary fibre and nonstarch carbohydrates in wheat and barley whole meals.

Solid state fermentation has been previously used for detoxification and destruction of undesirable factors present in vegetable protein sources. Pal and Walia (2001) used Rhizopus oligosporus fermentation for rapeseed meal treatment and observed a reduction in CF as well as other antinutritional factors including glucosinolate, hiooxazolidones and phytic acid by 25.5, 43.1, 34, and 42.4%, respectively. Although the observed glucosinolate level reduction in FCM in the current study (38%) was lower than the report of Pal and Walia (2001), it is still advantageous to use L. salivarius over R. oligosporus as the later may increase the accumulation of undesirable end products such as aflatoxins (Mienda et al., 2011). Reduction in glucosinolates during fermentation may be due to the utilisation of glucose and sulphur moieties by microbial enzymes. This explanation is supported by observation of Verbiscar et al. (1981). The authors showed that after 21 days of jojoba meal fermentation by LAB at 26°C, total toxicants decreased by 95 to 98%. This was due to modification of cyano group by the nitrilase enzyme of LAB (Legras et al., (1990).

The results from the digestibility trial showed that the CP digestibility of FCM was higher (P<0.05) compared to CM, but not the AME (Table 3). Among the various AA, the enhancement of digestibility was observed for Met, Cys, Arg, Asp, Glu, and Ser. The observed protein quality might be due to the secretion of enzymes such as cellulase, phytase, and xylanase during the growth of microbes to convert the fibre materials for monosaccharide (Ugwuanyi et al., 2008). Some bacteria from the group of LAB, such as L. cellobiosus, P. pentosaceu, L. fermentum, L. brevis and L. plantarum, have been reported to have proteinase and aminopeptidase activities (Mugula et al., 2003). Pranoto et al. (2013) reported that, during fermentation, proteolysis could produce more peptides and AA and therefore increase digestible and soluble protein portion. In addition, some LAB such as L. plantarum have tannase activity (Duodu et al., 2003), breaking tannin complex with protein. Thereby, a combination of hydrolysis of protein matrix and releasing protein from complexes may attributed to LAB fermentation. The improvement in CP digestibility may also be attributed to the lower fibre content in FCM. This finding is supported by the reports of Siregar et al. (1982) and Brenes et al. (2002) that increase in dietary fibre or oligosaccharides reduced apparent protein digestibility in duck and chicken.

Table 1. Feed composition of experimental diets.

	Basal diet^°	Assay diets
		CM	FCM
Ingredients, %
Corn	54.97	-	-
Soybean meal (44%)	4.10	-	-
Palm oil	5.00	-	-
Corn gluten	1.00	-	-
Dicalcium phosphate	2.50	0.60	0.60
Calcium carbonate	0.50	0.60	0.60
Premix^#	1.00	1.00	1.00
Salt	0.30	0.30	0.30
L-lysine	0.30	-	-
Choline chloride	0.08	-	-
Sodium bicarbonate	0.05	-	-
DL-methionine	0.20	-	-
CM	30.00	97.00	97.00
Titanium dioxide	-	0.50	0.50
Calculated analysis
Metabolisable energy, MJ/kg	12.9	9.9	9.8
CP, %	20	39.9	40.9
L-lysine, %	1.0	1.6	1.6
DL-methionine, %	0.6	0.8	0.8
Methionine+cysteine, %	1.0	1.8	1.7
Threonine, %	0.8	1.7	1.7

CM, canola meal; FCM, fermented canola meal; CP, crude protein.

^°The basal diet was fed to all birds for 4 d before introduction of the assay diets.

^#Premix provided the following (per kilogram of diet): vitamin A, 2300 U; vitamin D₃, 400 U; vitamin E, 1.8 mg; vitamin B₁₂, 3.5 mg; riboflavin, 1.4 mg; panthotenic acid, 2 mg; nicotinic acid, 7 mg; pyridoxine, 0.25 mg; folic acid, 0.15 mg; menadione, 0.3 mg; thiamin, 0.15 mg; manganese oxide, 35 mg; ferrous sulfate, 35 mg; zinc oxide, 30 mg; copper sulfate, 60 mg; cobalt carbonate, 5 mg; potassium iodine, 0.6 mg; selenium vanadate, 0.09 mg.

Table 2. Composition of canola and fermented canola meals on a dry matter basis.

	CM	FCM	pooled SEM	P
Item
GE, MJ/kg	18.5	18.6	2.6	0.488
Ash, %	6.2	6.3	0.6	0.231
CP, %	41.2	42.2	0.3	0.013
CF, %	12.0	10.1	0.5	0.033
EE, %	3.3	3.5	0.4	0.641
Glucosinolate, µmol/g	22.0	13.6	1.7	0.010
AA
Aspartic acid	2.57	2.70	0.19	0.463
Serine	1.81	1.83	0.07	0.771
Glutamic acid	6.59	6.89	0.42	0.507
Glycine	2.13	2.15	0.08	0.905
Histidine	1.22	1.20	0.03	0.597
Arginine	2.54	2.50	0.09	0.756
Threonine	1.81	1.83	0.06	0.794
Alanine	1.59	1.66	0.10	0.550
Proline	2.37	2.45	0.13	0.591
Cysteine	1.07	1.04	0.61	0.597
Tyrosine	1.03	1.1	0.03	0.643
Valine	1.90	1.94	0.10	0.663
Methionine	0.77	0.76	0.05	0.830
Lysine	1.65	1.72	0.14	0.646
Isoleucine	1.35	1.39	0.07	0.616
Leucine	2.63	2.71	0.13	0.567
Pheylalanine	1.79	1.76	0.05	0.636
Tryptophan	0.39	0.39	0.01	0.561

CM, canola meal; FCM, fermented canola meal; GE, gross energy; CP, crude protein; CF, crude fibre; EE, ether extract; AA, amino acids.

Table 3. Apparent ileal digestibility of nutrients and amino acids of canola and fermented canola meals on a dry matter basis.

	CM	FCM	Pooled SEM	P
Item
AME, MJ/kg	10.24	10.13	31.99	0.451
CP, %	77.44	81.56	1.22	0.028
AA
Aspartic acid	74.6	79.7	0.81	0.003
Serine	72.5	78.6	2.11	0.045
Glutamic acid	81.2	83.9	0.81	0.030
Glycine	73.2	77.5	2.81	0.208
Histidine	77.7	82.1	3.24	0.246
Arginine	81.6	85.9	1.52	0.047
Threonine	68.5	75.0	3.42	0.129
Alanine	74.4	75.6	3.10	0.722
Proline	69.5	76.9	2.84	0.061
Cysteine	69.6	78.5	2.66	0.028
Tyrosine	73.0	80.0	2.63	0.058
Valine	72.1	76.3	2.98	0.240
Methionine	78.7	85.0	2.01	0.035
Lysine	76.2	79.4	2.12	0.201
Isoleucine	73.2	76.5	3.48	0.394
Leucine	76.4	79.3	2.77	0.366
Pheylalanine	76.6	81.3	2.67	0.153
Tryptophan	89.3	89.5	0.94	0.811

CM, canola meal; FCM, fermented canola meal; AME, apparent metabolisable energy; CP, crude protein; AA, amino acids.

Conclusions

Solid state fermentation of CM using L. salivarius reduces CF (by 16%) and glucosinolate content (by 38%), while enhances the CP and some essential AA. This treatment may improve the overall nutritive value of CM for broiler chickens.

Acknowledgements

the project was funded by the Malaysian Ministry of Education through the Long-Term Research Grant Scheme, Putrajaya, Malaysia, and the Al Ghurair Foods, Dubai, United Arab Emirates.