Substitution of crystalline l-lysine with l-lysine enriched fermentation broth in feed and effect on the performance of broiler chicks

Abstract

l-lysine is a limiting amino acid in cereal grains and a certain amount is essentially required to supplement the feed for optimum growth of poultry and pigs. The study was conducted to test performance of commercial broiler chicks fed on a feed supplemented with indigenously produced crude form of l-lysine (fermentation broth). A mutant of local strain of Corynebacterium glutamicum was used as source bacterium for lysine production on cane molasses based production medium. A total of 2500 one-day-old broiler chicks (mixed sex) were allocated at random to five dietary treatments (500 chicks/treatment). Basal diet provided 8 g total lysine/kg feed (group A). Groups B and C (positive control) were supplemented with 2 and 3 g crystalline lysine-HCl/kg feed respectively, whereas groups D and E were supplemented with lysine enriched fermentation supernatant to provide similar amount of lysine to that of groups B and C. Least significant difference test revealed highly significant (P<0.001) effect of two levels of fermentation product or crystalline lysine (HCl) on broiler performance. Significant improvement (P<0.05) in feed conversion ratio (FCR) was observed in treatment groups. Linear regression analysis (R ² values) revealed linear response of increasing lysine levels on growth and FCR from both lysine sources. Comparative analysis of antibody titres and histology of viscera revealed no toxic effect of the treatment. The results are suggestive of equal bioavailability, safety and efficacy of the test product in comparison to purified crystalline l-lysine (HCl).

Keywords:

• liquid lysine
• fermentation
• cane molasses
• broiler

Introduction

Need of l-lysine (lysine) as an essential amino acid for mono-gastric species is well known. Commercial broiler chicks require 11 g lysine/kg feed from 0 to 3 weeks of age and 10 g/kg feed from 3 to 6 weeks of age (NRC 1994) to show optimum weight gain/growth. Poultry and swine feed is formulated in such a way that available lysine from feed ingredients is calculated and formula requirement is then balanced through addition of crystalline lysine HCl. Anastassiadis (2007) estimated annual production of lysine around 8×10⁵ tones, majority of which was used as animal feed supplement. World demand of lysine is met through fermentative production (Kalinowski et al. 2003; Becker et al. 2011) from mutated strains of Corynebacterium glutamicum. On completion of fermentation process, lysine is purified in crystalline form (as HCl or sulphate salts) through ion exchange resin method. The process of down streaming is exhaustive and costly. However, products-like liquid l-lysine (LLB50^® from Ajinomoto-Eurolysine) and spray dried granules of l-lysine sulphate (Biolys60^® from Degussa) are now available in European market (Kircher and Pfefferle 2001) which do not apply ion exchange resin for purification. By use of these cheep forms of lysine in poultry feed on commercial basis, cost of production of poultry and pork meat could be reduced.

World broiler production was reported around 78,283 thousand tons in 2011, (Anonymous 2011). At least 0.1% crystalline lysine (HCl) is included in broiler feed to meet formula requirements. This means a reasonable demand of liquid lysine in market can also be explored. The present study was designed to evaluate possibility of production of liquid lysine from indigenous mutant strain of C. glutamicum and comparative in-vivo efficacy of this liquid lysine on performance of commercial broiler chicks. Safety of the liquid product was confirmed through histological examination of viscera, levels of antibody titres and mortality ratio among treatment groups.

Materials and methods

Bacterium, media and lysine production

A mutant of local strain of C. glutamicum was used for lysine production in 3 l jar fermentor on cane molasses based medium as described by Rehman et al. (2011).

Analytical methods

Lysine and total sugars were determined by the methods described by Hsieh et al. (1995) and Dubois et al. (1956), respectively.

Broiler feed

The basal diet provided 8 g of total lysine per kilogram (Table 1) and was fed to negative control group (A). The groups B and C (positive control) were supplemented with crystalline HCl salt of lysine at the rate of 2 and 3 g/kg feed, respectively. The treatment groups D and E were supplemented with fermentation broth at the rate of 80 ml/kg (equivalent to 2 g l-lysine) and 120 ml/kg (equivalent to 3 g l-lysine), respectively (Table 2). In groups C and E, lysine supplementation was reduced equivalent to 2 g/kg from fourth week onwards in order to match NRC (1994) recommendations during this period. One gram of lysine was analysed to be available from 40 ml of fermentation broth. Diet of group A provided 72.72%, groups B and D provided 90.9% and that of groups C and E provided 100% of NRC (1994) recommendations for first three weeks. The fermentation broth was mixed just before feeding in a 100 kg mixer, according to daily total feed requirement of each group. All feed refusal in the feeders were collected, weighed to calculate real intake and then feed conversion ratio (FCR) was calculated. Dead chicks were also weighed to make adjustment in FCR values.

Table 1. Ingredient and chemical composition of basal feed.

Ingredients	Rate of inclusion (kg/1000 kg)	Chemical composition^a	g/kg
Corn	398.7	Metaboliseable energy (MJ/kg)	11.42
Rice broken	100.0	Crude protein	193.1
Rice polish	100.0	Fat	34.1
Sunflower meal	24.6	Fibre	53.4
Soybean meal	200.0	Total ash	62.2
Canola meal	110.0	Salt	03.8
Limestone	12.7	Calcium	10.0
Bone	18.0	Phosphorus (total)	8.5
Molasses	30.0	Phosphorus (available)	4.5
Dl-Methionine	8.0	Lysine (total)	8.0
	5.0	Methionine	42.0
Vitamin premix		Aflatoxin^b (ppb)	30.77
		Sodium	0.5
		Potassium	8.7
^aCalculated.
^bAnalysed by AFLATEST® (VICAM, Watertown, MA, USA).

Treatment groups and management

Two thousand and five hundred commercial broiler chicks (Hubbard, day-old, mixed sex) were procured from a local hatchery and housed in a conventional commercial open house (natural ventilation and manual feeding and watering system). Five groups of 500 chicks each were made. All groups were further partitioned in five replicates of 100 chicks each at random and kept at standard management conditions. Chicks were vaccinated against New Castle Disease Virus (NDV) and Infectious Bronchitis Virus (IBV) just after arrival at farm with commercially available vaccine (ND + IB, Intervet^®), and a booster against NDV on 25th day of age.

Table 2. Detail of group.

Groups	Lysine added (g/kg feed)	Total lysine (g/kg feed)	Lysine sources	Percentage of NRC (1994) recommendations	Group definition
A	Nil	8	Basal feed	72.72	Negative control
B	2	10	Basal feed + crystalline l-lysine HCl	90.90	Positive control
C	3	11	Basal feed + crystalline l-lysine HCl	100	Positive control
D	2	10	Basal feed+fermentation broth	90.90	Treatment
E	3	11	Basal feed+fermentation broth	100	Treatment

Antibody titres

On 42nd day of age, blood was taken at random from five chicks from each replicate in 3 mL disposable syringes. Geometric mean titre (GMT) of antibodies against NDV was determined by hemagglutination inhibition test (HI) using four hemagglutinating units of locally produced ND antigen (Hussain et al. 1988, 1989).

Histology of viscera

Samples of liver and kidney (1×1cm) were fixed in 10% buffered formalin from slaughtered chicks at 42nd day, dehydrated in ascending grades of alcohol, cleared in xylene and embedded in paraffin blocks. Sections of 4–5 µm were cut and stained with Harris hematoxilin and eosin stain for intracellular examination (Humason 1972).

Statistical analysis

Means were subjected to one-way analysis of variance (ANOVA) and least significant difference (LSD) test, using statistical software SPSS 15.0 (2006). Effects of treatment were also evaluated through linear regression analysis (curve estimate) by comparing negative control with positive control groups and negative control with treatment groups.

Results

Lysine yield

Lysine was quantified to be 2.5 g per 100 ml of fermentation supernatant.

Total sugars in molasses

Total sugars per kilogram of cane molasses (supernatant) were quantified to be 483 g. Volume to weight ratio of molasses (supernatant) was 3 ml to 4 g.

Weight gain and FCR Performance after 3 and 6 weeks

Highly significant difference (P<0.001) in live body weight and FCR at 21st and 42nd day of age were observed when positive control groups (B and C) and treatment groups (D and E) were compared with negative control (group A). Weight gain of groups with highest lysine level (C and E) were significantly higher (P<0.001) than that of A, B and D. There was no significant difference among values of live body weight of positive control and treatment groups with similar lysine levels (B, D) and (C, E) at the end of 3rd and 6th week of age. Similarly, no significant difference was observed in FCR values of positive control and treatment groups with similar lysine levels (B, D) and (C, E) at 21st and 42nd day of age. There was also no significant difference in mortality among all experimental groups when compared at 21st and 42nd day of age (Table 3). Linear regression analysis revealed that effect of supplemented lysine from either source was highly significant on live body weight and FCR when compared at 21st day and 42nd of age. R ² values revealed linear effect of lysine on live weight gain and FCR after 3 and 6 weeks of feeding in both the positive control and treatment groups (Table 4).

Table 3. Comparative effect of lysine sources on body weight, feed conversion ratio (FCR) and mortality of broiler chicks.

		Body weight (kg/chick)		FCR (kg/kg)		Mortality (%)
Groups	Total lysine (g/kg feed)	21 day mean±SD	42 day mean±SD	21 day mean ±SD	42 day mean ±SD	21 day mean±SD	42 day mean±SD
A	8	0.506^d±0.008	1.675 ^d±0.021	1.954^a±0.313	2.546^a±0.049	3.2±1.64	6.0 ±2.24
B	11	0.583^b±0.011	1.765^bc±0.065	1.692^b±0.037	2.322^b±0.06	4.2±2.17	7.4±1.52
C	12	0.636^a±0.009	1.98^a±0.028	1.558^c±0.026	2.05^c±0.028	2.6±0.55	5.8±0.84
D	11	0.574^c±0.008	1.814^b±0.33	1.724^b±0.015	2.24^b±0.081	3.0±1.73	6.0±2.24
E	12	0.632^a±0.008	1.99^a±0.021	1.564^c±0.023	2.038^c±0.02	2.8±0.45	6.0±0.71
Note: A, basal feed; B, basal feed + crystalline l-lysine HCl (2 g/kg feed); C, basal feed + crystalline l-lysine HCl (3g/kg feed); D, basal feed + fermentation broth equivalent to 2 g lysine/kg feed; E, basal feed+fermentation broth equivalent to 3 g lysine/kg feed.
n=5 (pens of 100 chicks each) per group
^a–dMeans in a column lacking common superscript differ significantly (P≤0.001), LSD.

Table 4. Linear regression (curve estimate) analysis comparing negative control with positive control groups and negative control with treatment groups (P < 0.001).

	Groups compared
	A, B and C				A, D and F
Statistical parameters	BW 21 days	BW 42 days	FCR 21 days	FCR 42 days	BW 21 days	BW 42 days	FCR 21 days	FCR 42 days
R ^2	0.908	0.603	0.938	0.787	0.928	0.86	0.952	0.921
Adjusted R ^2	0.901	0.572	0.934	0.77	0.922	0.849	0.948	0.915
F	128.01	19.71	197.86	47.913	167.375	79.832	257.736	151.336
y=a+b (x)	0.2957+0.268x	0.594+1.189x	−0.927+2.699x	−1.054+3.402x	0.294+0.266x	0.705+1.097x	−0.927+2.705x	−1.212+3.527x
P	>0.001	=0.001	>0.001	>0.001	>0.001	>0.001	>0.001	>0.001
A, negative control; B, positive control (crystalline l-lysine HCl (2 g/kg feed); C, positive control (crystalline l-lysine HCl (3 g/kg lysine), D, fermentation broth equivalent to 2 g lysine/kg feed; E, basal feed ingredients+fermentation broth (3 g/kg lysine); BW, body weight.

Antibodies response, gross and microscopic examination of organs

Analysis of variance showed no significant difference in values of geometrical mean titres (GMT) of antibodies against NDV, among all groups (P<0.05; Table 5).

Table 5. Geometrical mean titres (GMT) of antibodies against new castle disease virus on day 42.

			Distribution of titre in tested chicks
Group	Replicate	No. of chicks tested	1:8	1:16	1:32	1:64	1:128	1:256	GMT	SEM
A	A1	5	–	1	1	1	2	–	54.95
	A2	5	–	2	2	–	1	–	28.84
	A3	5	–	2	–	–	3	–	50.71
	A4	5	1	–	–	4	–	–	42.13
	A5	5	–	–	–	4	1	–	72.44
Total		25	1	5	3	9	7	–	49.89	7.21
B	B1	5	–	–	2	2	1	–	55.72
	B2	5	–	1	1	2	–	1	55.72
	B3	5	1	–	2	1	1	–	36.31
	B4	5	–	1	1	2	1	–	47.86
	B5	5	–	1	1	2	–	1	55.72
Total		25	1	3	7	9	3	2	50.27	3.81
C	C1	5	–	–	–	3	1	1	97.05
	C2	5	–	1	2	2	–	–	36.31
	C3	5	–	1	1	3	–	–	41.69
	C4	5	–	2	1	1	1	–	36.31
	C5	5	–	–	2	2	1	–	56.24
Total		25		4	6	11	3	1	53.52	11.48
D	D1	5	–	–	2	2	1	–	55.72
	D2	5	–	–	–	3	1	1	97.05
	D3	5	1	–	2	1	1	–	36.31
	D4	5	–	1	1	2	1	–	47.86
	D5	5	–	1	1	3	–	–	41.69
Total		25	1	2	6	11	4	1	55.73	10.83
E	E1	5	–	–	2	2	1	–	55.72
	E2	5	–	–	3	2	–	–	41.69
	E3	5	–	–	2	2	1	–	55.72
	E4	5	–	–	–	4	1	–	72.44
	E5	5	–	–	2	3	–	–	47.86
Total		25	–	–	9	13	3	–	54.69	5.16
F	F1	5	–	1	1	2	–	1	55.72
	F2	5	–	–	2	2	1	–	55.72
	F3	5	–	1	1	2	1	–	47.86
	F4	5	1	–	–	–	4	–	73.52
	F5	5	–	2	2	–	1	–	28.84
Total		25	1	4	6	6	7	1	52.33	7.23
aA, basal feed; B, basal feed + crystalline l-lysine HCl (2 g/kg feed); C, basal feed + crystalline l-lysine HCl (3 g/kg feed); D, basal feed + fermentation broth equivalent to 2 g lysine /kg feed; E, basal feed ingredients + fermentation broth equivalent to 3 g lysine /kg feed.

Gross examination of all visceral organs revealed normal color, size and consistency. Histology of liver tissues showed normal hepatic triad with no lesions of degeneration or inflammatory reaction. Similarly, kidney tissues showed normal glomerular system.

Discussion

Present study was conducted to estimate in-vivo safety and efficacy of locally produced l-lysine (HCl) in crude form (without purification through ion exchange process). All treatment groups provided either lower lysine levels than recommended or equal to recommendation for commercial broilers by NRC (1994). This design is in line to test specific effect of lysine on growth and FCR as proposed by Batterham (1992). No significant difference among FCR of the treatment groups (with similar levels of lysine from liquid or crystalline source) along with no pathological visceral lesions and protective antibody titres are reflective of the safety and efficacy of liquid lysine and results are consistent with the findings of Emmert et al. (1999) on bioavailability of liquid lysine in broiler chicks. Another successful process demonstration of production of granular lysine sulphate at Degussa-Huls AG, Germany was reported by Kircher and Pfefferle (2001). The fermentation broth was concentrated and passed through spray granulator. The results showed that the product was as good as crystalline l-lysine-HCl in terms of feed intake, weight gain and feed utilisation of piglets. The results of the present study regarding product safety and FCR of treatment groups are in agreement with the findings of Kircher and Pfefferle (2001). However, in this study, lysine product was used in liquid form with out biomass.

Peisker (1998) tested stability of liquid lysine in feed processing at 90, 110 and 130°C in a broiler growth trial. FCR was reflective of a high level of lysine efficiency. The author also concluded that addition of molasses to the diet did not decrease availability of dietary lysine. In the present study, liquid lysine in molasses was exposed to 80°C for half an hour only for inactivation of biomass. However, findings of Peisker (1998) favour the possibility to concentrate our product at high temperature without loss of efficacy. Statistical analysis showed that significant difference in FCR was due to increasing lysine levels at both 21st and 42nd day of age. Some studies (Baker 1996; Schutte and Smink 1998) suggest a bit higher total lysine requirement for broilers than NRC (1994) recommendation. Regression analysis on weight gain and feed efficiency after 21 and 42 days of feeding showed linear response to increasing levels of lysine from either source (crystalline or liquid). However, dose requirement calculations were beyond the scope of this study and treatment groups were only compared with negative and positive control groups to verify in-vivo efficacy and safety of the test product. Broiler performance gained in this study was lower than standards even at recommended NRC (1994) standard (11 g/kg total lysine). This is due to low energy and low crude protein levels in basal diet used in this experiment. These levels were kept on lower side to clearly define the effect of treatment. Poor performance at low dietary protein level is in agreement with Jiang et al. (2005), who reported failure of chicks to overcome protein deficiency despite increasing levels of essential amino acids up to 110% of NRC (1994) and lysine up to 120% of NRC (1994) recommendations.

The NDV-HI titres of 1:8 and above were considered as indicative of specific and protective immunity (Allan and Gough 1974; Shuaib et al. 2006). Titres well above this level (1:8) were found in all treatment groups. Normal histology of viscera is also suggestive of the safety of the product.

Conclusions

Lysine enriched cane-molasses can be used safely in poultry feed as lysine source and it can be substituted with cane-molasses in mash type feed which is used routinely to overcome dusting problem. The product may also be dried through spray granulation for addition in crumbs type commercial feed.

Acknowledgements

Financial support from Higher Education Commission of Pakistan is highly appreciated.