Effects of citric acid residue and lactic acid bacteria on fermentation quality and aerobic stability of alfalfa silage

Figures & data

Table 1. Chemical and microbial composition of fresh alfalfa.

Items	Alfalfa
Dry matter, g/kg FW	205
Crude protein, g/kg DM	230
Water soluble carbohydrate, g/kg DM	63.7
Buffering capacity, mEq /kg DM	215
Neutral detergent fibre, g/kg DM	402
Acid detergent fibre, g/kg DM	275
Lactic acid bacteria, log cfu/g FW	6.40
Enterobacteriaceae, log cfu/g FW	6.85
Yeasts and moulds, log cfu/g FW	6.55

FW: fresh weight; DM: dry matter; cfu: colony-forming units.

Data are presented as means of five replicates.

Table 2. Effect of additives on pH value, dry matter, ammonia nitrogen, water soluble carbohydrate contents and microbial compositions of alfalfa during ensiling.

Items	Ensiling days	Treatments				SEM	p Value
		CON	L	C	CL		T	D	T × D
Dry matter, g/kg FW	3	202	206	204	214	1.01	<0.001	ns	ns
	6	197^b	199^b	221^a	207^ab
	9	192^b	206^a	207^a	210^a
	15	195^b	204 ^ab	203^ab	211^a
	30	197^b	204^ab	206^ab	213^a
	45	194^b	195^b	202^a	207^a
pH value	3	5.30^a	5.11^a	5.19^a	4.54^c	0.035	<0.001	<0.001	<.001
	6	4.99^a	4.93^a	4.74^b	4.35^c
	9	5.03^a	4.87^a	4.57^b	4.39^b
	15	5.00^a	4.85^b	4.45^c	4.32^c
	30	5.01^a	4.73^b	4.40^b	4.47^b
	45	4.86^a	4.89^a	4.32^b	4.38^b
NH3-N, g/kg TN	3	44.7^a	45.9^a	25.6^b	18.7^c	4.03	<0.001	<0.001	<.001
	6	49.9^a	50.5^a	28.2^c	33.2^b
	9	49.9^a	52.2^a	32.3^b	35.1^b
	15	72.4^a	52.1^b	32.4^c	59.2^b
	30	76.6^a	76.5^a	32.1^c	65.4^b
	45	106^a	102^a	82.1^b	78.4^c
Water soluble carbohydrate, g/kg DM	3	21.3^b	18.3^b	45.4^a	17.4^b	1.22	<0.001	<0.001	<.001
	6	16.7^b	13.6^c	39.5^a	15.2^bc
	9	12.4^b	11.3^bc	30.7^a	9.62^c
	15	7.17^b	8.49^b	13.2^a	7.73^b
	30	5.70^c	6.65^c	10.5^a	7.83^b
	45	5.93^b	6.12^b	8.92^a	6.57^b
Lactic acid bacteria, log cfu/g FW	3	7.75	7.95	7.76	7.96	0.095	0.011	<0.001	<.001
	6	9.30	9.84	9.05	9.12
	9	9.86^b	10.1^a	9.05^c	9.96^ab
	15	9.10^c	9.22^bc	9.78^a	9.46^ab
	30	9.12	9.22	8.61	8.94
	45	8.10^a	7.59^b	7.89^ab	8.11^a
Enterobacteriaceae, log cfu/g FW	3	6.98^a	6.02^b	5.17^c	4.80^d	0.173	<0.001	<0.001	<.001
	6	6.24^a	4.65^b	3.85^c	3.86^c
	9	6.00^a	4.08^b	3.61^c	3.22^c
	15	5.36^a	2.90^b	2.72^b	2.38^c
	30	5.27^a	2.71^b	2.57^b	2.12^c
	45	4.59^a	2.88^b	2.31^c	1.87^c

FW: fresh weight; DM: dry matter; cfu: colony-forming units; CON: control; L: 1 × 10⁶ log cfu/g lactic acid bacteria inoculant (Lactobacillus plantarum + Lactobacillus buchneri); C: 12 g/kg citric acid residue; CL: C + L; D: effect of ensiling days; T: effect of treatments; T × D: effect of treatment and ensiling days interactions; ns: non-significant; SEM: standard error of means.

Data are presented as means of five replicates. Values in the same row with different superscripts are significantly different (p < .05).

Table 3. Effect of additives on fermentation acids of alfalfa during ensiling.

Items	Ensiling days (D)	Treatments				SEM	p Value
		CON	L	C	CL		T	D	T × D
Lactic acid, g/kg DM	3	14.7^a	17.1^a	8.06^b	13.4^a	4.31	<0.001	<0.001	<.001
	6	21.6^a	23.1^a	12.7^b	19.7^a
	9	44.8^a	46.3^a	15.5^c	23.1^b
	15	49.0^a	47.9^b	24.1^c	36.5^b
	30	61.9^c	117^a	57.3^c	102^b
	45	101^b	87.1^c	110.5^a	116^a
Acetic acid, g/kg DM	3	8.05^a	7.76^a	4.97^b	5.41^b	1.24	<0.001	<0.001	<.001
	6	6.33^a	6.07^a	6.57^a	4.84^b
	9	15.2^a	8.42^b	8.32^b	6.37^c
	15	17.4^a	9.67^b	8.65^bc	7.21^c
	30	17.4^b	37.1^a	14.5^b	16.9^b
	45	34.6^b	39.5^a	24.6^c	26.6^c
Lactic acid/acetic acid	3	1.84^bc	2.22^ab	1.61^c	2.46^a	0.146	<0.001	<0.001	<.001
	6	3.41^b	3.83^ab	1.96^c	4.06^a
	9	2.95^c	5.51^a	1.87^d	3.63^b
	15	2.82^b	4.98^a	2.79^b	5.06^a
	30	3.57^b	3.17^b	3.95^b	6.08^a
	45	2.91^b	2.20^c	4.49^a	4.38^a
Propionic acid, g/kg DM	3	0.78^c	2.23^a	0.73^c	1.60^b	0.080	<0.001	<0.001	<.001
	6	1.11^a	1.31^a	0.78^b	1.10^a
	9	1.03	1.48	1.26	1.36
	15	1.38^b	2.44^a	0.80^b	2.30^a
	30	0.94^b	0.85^b	1.26^b	2.60^a
	45	1.28^b	2.19^a	0.72^b	2.73^a
Butyric acid, g/kg DM	3	2.24	ND	ND	ND	0.112	<0.001	<0.001	<.001
	6	1.74	1.59	ND	1.80
	9	ND	ND	ND	ND
	15	1.94	1.75	1.89	1.65
	30	2.34	ND	1.92	ND
	45	1.89	1.68	1.77	1.61

FW: fresh weight; DM: dry matter; cfu: colony-forming units; CON: control; L: 1 × 10⁶ log cfu/g lactic acid bacteria inoculant (Lactobacillus plantarum + Lactobacillus buchneri); C: 12 g/kg citric acid residue; CL: C + L; D: effect of ensiling days; T: effect of treatments; T × D: effect of treatment and ensiling days interactions; SEM: standard error of means; ND: No detected.

Data are presented as means of five replicates. Values in the same row with different superscripts are significantly different (p < .05).

Table 4. Effect of additives on structural carbohydrates composition and aerobic stability of alfalfa ensiled for 45 days.

Items	Treatments				SEM	p Value
	CON	L	C	CL
NDF, g/kg DM	325^a	327^a	314^ab	302^b	3.59	.019
ADF, g/kg DM	230^a	226^ab	229^a	216^b	2.38	.107
ADL, g/kg DM	49.6	48.7	50.9	53.5	0.986	.375
Hemicellulose, g/kg DM	95.2^ab	101^a	84.7^b	86.4^b	2.59	.054
Cellulose, g/kg DM	180^a	177^a	178^a	162^b	2.75	.043
Aerobic stability, h	115^c	132^b	84.0^d	144^a	6.82	<.001

DM: dry matter; NDF: neutral detergent fibre; ADF: acid detergent fibre; ADL: acid detergent lignin; CON: control; L: 1 × 10⁶ log cfu/g lactic acid bacteria inoculant (Lactobacillus plantarum + Lactobacillus buchneri); C: 12 g/kg citric acid residue; CL: C + L; SEM: standard error of means.

Data are presented as means of five replicates. Values in the same row with different superscripts are significantly different (p < .05).

Figure 1. The change of (a) pH, (b) water soluble carbohydrate, (c) ammonia nitrogen (NH₃-N), (d) lactic acid, (e) acetic acid and (f) yeasts and moulds of alfalfa silages during aerobic exposure. FW: fresh weight; DM: dry matter; NH₃-N: ammonia nitrogen; TN: total nitrogen; cfu: colony-forming units; CON: control; L: 1 × 10⁶ log cfu/g lactic acid bacteria inoculant (Lactobacillus plantarum + Lactobacillus buchneri); C: 12 g/kg citric acid residue; CL: C + L. Data are presented as means of five replicates. T: effect of treatments; D: effect of days of air exposure; T × D: interaction between treatments and days of exposure (p<.05).