Effect of different particle lengths on the bacterial population, fermentation profiles and nutritive value of whole maize plant silage

The effect of particle length (2, 4 and 6 cm) on the quality of whole maize plant silage was evaluated by determining the bacterial densities, fermentation characteristics, proximate composition and in situ degradation rates over a 5-week period of ensiling.

Higher populations of TVB and LAB were detected in maize silage chopped to 2cm particle length compared with 4 or 6cm. length. Lactic acid levels were higher, and those of butyric acid lower, in the 2cm particle length maize silage. Percentages of DM, OM and CP were higher in the 2 cm length treatment as were the rates of degradation of DM in in situ rumen incubation. levels of NDF and ADF were lower in the 2cm chop silage.

Key words: fermentation characteristics, in situ technique, LAB, lactic acid, VFA

Introduction

Ensiling is a process of forage preservation method of conserving the nutrient content of forage, ensuring a good nutritional value when used as feed (Vervaeren et al 2010). It is aimed for long term storage of the feed and constraints of feed supply from seasons can be eliminated (Yan Ping et al 2011). Ensiling is a process of converting the anaerobic fermentation of water-soluble carbohydrates into lactic acid and volatile fatty acids (VFA) by help of anaerobic and lactic acid bacteria (LAB). Silage fermentation characteristics and nutritive value of ensilaged mass can be influenced by many factors. The particle length of the plant material can also contribute in determining the quality of the silage via packing density which indirectly affects the silage fermentation quality. Moreover, the use of short particle length provides more surface areas for the attachment of bacteria during ensiling process. Quality of the silage is achieved when lactic acid is sufficiently produced. It is the predominant and the most efficient fermentation acid produced by lactic acid bacteria which will rapidly reduce the pH of the silage. The faster the fermentation process is completed, the more nutrients will be retained in the silage (Schroeder 2012). As a result, when the pH decreases, it helps in preventing detrimental anaerobes bacteria from multiply, thus preserving the nutritional values and palatability of the moist forage (Yan Ping et al 2011). Maize silage has been used as a main crop for ensiling due to its best nutritional quality and excellent ensiling properties (Allen et al 2003). During these days, nutritionists, agronomists, and dairy producers have placed increased emphasis on factors affecting the nutritive value of whole maize plant silage (Bal et al 2000). Most studies were focussed on comparing the effect of mechanical processing on the cutting length and its effect on the fermentation characteristics and DM disappearance of maize silage. Also, the differences within the length of particle in those reported studies were too narrow (Andrae et al 2001, Johnson et al 2003a). To our best knowledge, little information is available on the existence of bacteria population that are directly affected by the different cutting length. Therefore, the aim of the current study was to determine the bacterial population, fermentation characteristics and nutritive value of whole maize plant silage prepared in different particle length.

Methods

Silage preparation and sampling

The whole maize plant (Suwan maize variety) used for making silage was harvested at dent stage of maturity around 11 weeks (24% DM) and cut into different particle length; 2cm, 4cm and 6cm. Each samples length was inserted into small air tight containers and prepared in triplicates. The air-tight containers were stored at room temperature and were opened for analysis on weekly basis starting from week 1 until week 5 of ensiling period. The content of these containers were thoroughly mixed and the samples were collected for determination of microbial analyses, pH, fermentation characteristics, chemical analysis and in situ DM, OM and CP disappearance and degradability.

Microbial analysis

The population of total viable bacteria (TVB) and lactic acid bacteria (LAB) isolated from the maize silage were determined by carrying out a 10-fold serial dilution of bacterial culture using sterile peptone water. A total of 0.1 mL aliquots of diluted bacterial suspension were spread on nutrient agar and MRS agar. The number of the colony forming units (CFU) was enumerated and recorded after 48 h of incubation at 37°C. The CFU per gram of the sample was calculated according to Thu et al (2011).

pH and fermentation analysis

Twenty five grams of fresh maize silage were mixed with 100 ml of distilled water and blended for 30 seconds in a blender. This was filtered through Whatman filter paper (No. 1) to obtain the extracts (Filya 2003). Immediately after extraction, the pH was measured using digital pH meter. The remaining filtrate was preserved with a few drops of 5% sulphuric acid and kept frozen at -20 ̊ C for lactic acid and VFA analysis. Lactic acid and VFA concentrations in the silage extract were determined using gas chromatography (Agilent 69890N Series Gas Chromatography System from Agilent Technologies, USA) equipped with a flame ionization detector.

Chemical analysis

For chemical analysis, dry matter (DM), organic matter (OM) and crude protein (CP) of maize silage were determined according to the procedure of (AOAC 1990); neutral detergent fiber (NDF), acid detergent fiber (ADF) were determined using methods previously described by Van-Soest et al (1991).

In situ rumen degradability study

In situ ruminal degradability was determined by incubating nylon bags containing whole maize plant silage samples into three ruminally - fistulated goats. A bag size of 13.5 cm and 8.5 cm, with pore size of 50 µm was used. The sample used for in situ study was taken from the week 5 ensiled maize. Samples of different particle lengths were dried and ground using 2 mm sieve. Later approximately 4 g of each sample was inserted into the nylon bags and placed in the rumen for 3, 6, 12, 24, 48, 72 and 96 h of incubation. For each incubation time, ruminal disappearance (%) of DM, OM and CP were calculated from the proportion remaining in the bags. Degradation kinetics of DM, OM and CP were calculated using the equation of Ørskov and McDonald (1979).

Statistical analysis

All data were statistically analyzed by using two-way and one-way Analysis of Variance (ANOVA); statistical significance among the treatment means was determined by Duncan’s multiple range tests. These analyses were performed by ANOVA using the General Linear Model (GLM) procedure of SAS package version 9.2.

Results and Discussion

The numbers of TVB and LAB colonies in the 2 cm particle length in the current study were higher than in the other two particles lengths at the end of first week of ensiling (Tables 1 and 2). This is because short particle length provides more surface area for the attachment of LAB during the ensiling process (Thu et al 2011). Another reason is that,the use of long particle length provides lots of air pockets between the particles. This condition prolongs the depletion of the oxygen and the fermentation of water-soluble carbohydrate in the container (Seglar 2003). In the present study, low numbers of bacterial colonies were detected in the 2 cm particle length after the second week of ensiling process. It is believed due to completion of the fermentation process at the end of week 1 which resulted from high population of bacteria detected in the same week that help to speed-up the fermentation of water-soluble carbohydrate.

Table 1. Total viable bacteria (TVB) colonies in whole plant maize silage with different particle length
Items	Particle length			SEM	p
Items	2 cm	4 cm	6 cm	SEM	p
Week 1	10.5^a	10.3^b	10.0^c	0.02	<0.0001
Week 2	9.14^b	9.58^a	9.64 ^a	0.08	0.0004
Week 3	8.72 ^b	9.13^a	9.16 ^a	0.18	0.0432
Week 4	8.44^a	8.79^a	8.62^a	0.27	0.35
Week 5	8.24^a	8.43^a	8.50^a	0.30	0.65
^abc Means without common superscript in each row differ at p<0.05

Table 2. Lactic acid bacteria (LAB) colonies in whole plant maize silage with different particle length
Items	Particle length			SEM	p
Items	2 cm	4 cm	6 cm	SEM	p
Week 1	10.6 ^a	10.0^b	9.20 ^c	0.12	<0.0001
Week 2	8.69^c	9.48^a	9.21^b	0.13	<0.009
Week 3	8.42 ^b	8.50^b	8.95^a	0.18	0.024
Week 4	8.20	8.62	8.56	0.31	0.28
Week 5	8.00	8.34	8.33	0.21	0.15
^abc Means without common superscript in each row differ at p<0.05

Woolford (1985) showed that LAB can grow together with aerobic bacteria in the presence of oxygen between the plant particles. Then fermentation was promoted by creation of an anaerobic environment and, population of LAB appeared predominately. There was a significant interaction observed between the length of particle and ensiling period for both TVB and LAB population from the whole maize plant silage (Table 3).

Table 3. Interaction between particle length and ensiling period of whole maize plant silage
log₁₀CFU/g	Particle length (PL)			SEM	PL	Week	PL x Week
log₁₀CFU/g	2 cm	4 cm	6 cm	SEM	PL	Week	PL x Week
TVB	9.06^b	9.25^a	9.13^ab	0.20	0.052	<0.0001	0.012
LAB	8.78^b	9.00^a	8.85^b	0.20	0.0035	<0.0001	<0.0001
pH	4.06	4.14	4.17	0.04	0.13	0.002	0.98
^abc Means without common superscript in each row differ at p<0.05

The pH values in the silage with different particle length did not differ throughout the five weeks. (Table 4). After two weeks of ensiling, all silages were found to be well preserved because the pH values were within the typical range 3.8-4.2. During the first week of ensiling the pH value was higher in the 2 cm length silage than in the other two. This could be attributed to the fact that the bacteria during this phase tend to be inefficient fermenters as they produce relatively low level of acid in exchange for the nutrient losses (Seglar 2003). After a week, the levels of pH steadily declined because the emergence of bacteria populations in this phase creates a faster rate of fermentation, thus conserving more nutrients such as water-soluble carbohydrate, peptides and amino acids. The fermentation process continues until the levels of pH are sufficiently low to inhibit, but not destroy, the growth potential of all organisms (Seglar 2003). The pH value for the 2 cm particle length silage tended (p=0.13) to be lower than for the silage with 4 and 6 cm particle length.

Table 4. Changes in pH during the fermentation of whole maize silage with different particle length
	Particle length			SEM	p
	2 cm	4 cm	6 cm	SEM	p
Week 1	4.26	4.32	4.39	0.11	0.11
Week 2	4.10	4.12	4.15	0.15	0.93
Week 3	4.07	4.10	4.12	0.16	0.92
Week 4	3.90	4.09	4.10	0.24	0.55
Week 5	3.95	4.05	4.10	0.09	0.23

High concentration of lactic acid detected in the 2 cm particle length silage (Table 5) was due to the fact that it was more densely packed than 4 cm and 6 cm particle length silage. This resulted in rapid change from aerobic to anaerobic conditions in the container that favours the LAB population to predominate. A high LAB population will produce larger amount of lactic acid. High quality silage is likely to be achieved when lactic acid is produced, as it is the most efficient fermentation acid, and reduces silage pH more efficiently than other fermentation products (McDonald et al 2002). Lactic acid is stronger than the other fermentation acids in silage (acetic, propionic, and butyric), and therefore inhibits the activity of other types of fermentative bacteria.

Apart from that, high concentration of acetic acid recorded in the 2 cm particle length in the current study during the first week of ensilation was due to fact that less time are required to achieve anaerobic environment stability. Unlike the 2 cm, the low concentration of acetic acids detected in the 4 and 6 cm particle length were manifested by slow turnovers of aerobic into anaerobic conditions due to large air pockets present in the container which in turn reduced the number of LAB production. Fermentation starts when anaerobic environment is stable and this initiates the growth of acetic acid producing bacteria. This type of bacteria has the ability to ferment soluble carbohydrates into acetic acid which keeps in reducing the pH and to set up the following fermentation phase (Schroeder 2012).

Table 5. Fermentation characteristics of maize silage
Items (%DM)	Particle length				SEM	p
	2 cm		4 cm	6 cm
Lactic
Week 1	4.62^a		4.30^b	3.63^c	0.21		0.0003
Week 2	5.79^a		5.22^ab	4.39^b	0.24		0.0094
Week 3	6.36^a		5.54^ab	5.43^b	0.51		0.0011
Week 4	6.69		6.70	6.20	0.50		0.67
Week 5	6.78		6.71	6.54	0.56		0.66
Acetic
Week 1	2.74^a		2.04^ab	1.78^b	0.25		0.011
Week 2	1.84^b		2.58 ^a	2.44 ^a	0.43		0.005
Week 3	1.75^ab		2.32 ^a	2.17^a	0.18		0.045
Week 4	1.54		1.48	1.47	0.32		0.063
Week 5	1.51		1.67	1.43	0.21		0.073
Propionic
Week 1	0.10		ND	ND	-
Week 2	ND		ND	0.01	-
Week 3	0.06		0.14	ND	-
Week 4	ND		0.08	0.06	-
Week 5	0.11		0.13	0.06	0.01		0.13
Butyric
Week 1		0.10 ^b	0.13^b	0.32^a	0.01		0.043
Week 2	0.07		0.09	0.11	0.15		0.82
Week 3	0.04		0.06	0.07	0.01		0.075
Week 4	0.01		0.06	0.07	0.001		0.364
Week 5	0.01		0.01	0.02	0.001		0.180
^{a, b, c} Means with different superscript in each row differ at p <0.05 ND = not detected

After week 4 and 5 of the ensiling period, the acetic acid concentration did not differ among the treatment groups. Our data are in agreement with the report of Johnson (2003a) that high levels of acetic acid result at 2, 3 and 6 days after ensiling of maize. However, the concentration of acetic acid was found to be similar between treatment groups after 57 days of ensiling. The concentration of propionic acid was low and did not appear to differ among all particle lengths. Propionate is seldom detected in well fermented silages because propionic-acid bacteria are very sensitive to low pH (Kung 2008). The concentration of butyric acid in 6 cm particle length silage was higher during week 1 of ensiling compared to the other two particle lengths. Chopping forages too long makes it difficult to pack the forages into the container causing air to remain trapped in the silage (Schroeder 2012). This factor contributes to emergence of spoilage microorganisms as a result of the slow change of the environment inside the silo from aerobic to anaerobic. Significant interactions were observed between the length of particle and ensiling period for lactic and acetic acid concentration. However, no interaction was observed in the propionate and butyrate acid production (Table 6).

Table 6. Interaction between particle length and ensiling period of fermentation acid of whole maize plant silage
% in DM	Particle length			SEM	PL	Week	PL x Week
% in DM	2 cm	4 cm	6 cm	SEM	PL	Week	PL x Week
Lactate	6.05^a	5.69^b	5.23^c	0.35	<0.0001	<0.0001	0.0003
Acetate	1.88	2.01	1.86	0.18	0.15	0.031	0.045
Butyrate	0.04^b	0.06^b	0.19^a	0.02	0.004	0.002	0.69
^{a, b, and c} Means with different superscript in each row differed at p<0.05

The percentages of DM, OM and CP in the 2 cm particle length silage were higher, and NDF and ADF lower, than in the 4 cm and 6 cm particle length silage (Table 7), however, the differences were relatively small.

Table 7. Effect of particle length on chemical composition of whole maize plant silage
	Particle length (PL)			SEM	PL	Week	PL × Week
	2 cm	4 cm	6 cm	SEM	PL	Week	PL × Week
DM,%	23.3^a	23.0^b	22.8^c	0.02	<0.0001	<0.0001	<0.0001
		% in DM
OM	96.2^a	95.9^b	95.8^c	0.34	<0.0001	<0.0001	<0.0001
CP	7.52^a	7.22^b	6.84^c	0.20	<0.0001	<0.0001	0.853
NDF	64.4^a	66.1^b	66.9^b	0.48	0.042	0.0013	0.067
ADF	35.6^b	36.1^ab	36.7^a	0.32	0.006	<0.0001	<0.0001
^abc Means without common superscript in each row differ at p<0.05

The higher values for potentially degradable DM and OM in the silage with 2cm length particles (Table 8) were due mainly to the increase in the soluble fraction (a). Bal (2000) and Johnson (2003b) also reported that shorter cutting length had a greater rate of DM disappearance of maize silage in a rumen incubation than with a longer cutting length. This difference could be related to higher availability of nutrients that are more digestible especially those derived from the starch in the grain portion of whole plant maize silage (Mehmet 2005). It is to be expected that the maize silage with lower conent of NDF and ADF (as in the 2cm length treatment) would have rate of DM disappearance as in the report by Johnson (2003b). These authors suggested that it could be attributed to the structure and solubility characteristics of the short particle length silage which would facilitate attachment of rumen microorganisms.

Table 8. Degradation of respective particle length of maize silage in the rumen of fistulated goats.
Items	Particle length			SEM	p
Items	2 cm	4 cm	6 cm	SEM	p
DM
a	20.7 ^a	18.3 ^b	15.7 ^c	0.55	0.0043
b	57.0 ^b	56.3 ^b	60.0 ^a	0.32	0.0112
c h^-1	0.058	0.056	0.055	0.002	0.135
PD	63.7 ^a	60.3 ^b	59.0 ^b	0.45	0.0007
OM
a	20.7 ^a	18.7 ^b	14.7^c	0.25	<.0001
b	57.0 ^b	56.3 ^b	60.0 ^a	0.31	0.012
c h^-1	0.058	0.056	0.055	0.001	0.57
PD	63.7 ^a	60.3 ^b	59.0 ^b	0.33	0.0007
CP
a	65.7 ^a	63.8 ^a	59.3^b	0.40	0.0008
b	23.8	23.6	26.7	0.38	0.091
c h^-1	0.06	0.06	0.058	0.002	0.443
PD	83.8 ^a	81.8 ^b	79.2^c	0.29	0.0013
^abc Means without common superscript in each row differ at p<0.05 a: rapidly degradable fraction; b: the slowly degradable fraction c: degradation rate; PD: potentially degradable fractions

Conclusions

Acknowledgements

The authors are grateful to the South East Asia Scholarship Organization (SEMEO-SEARCA) and ABI R&D Initiative Fund Project (10-05-ABI-AB035) for financial support.

References

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Effect of different particle lengths on the bacterial population, fermentation profiles and nutritive value of whole maize plant silage

K T Khaing, T C Loh², S Ghizan¹, R A Halim¹ and A A Samsudin²

Abstract

Introduction

Methods

Silage preparation and sampling

Microbial analysis

pH and fermentation analysis

Chemical analysis

In situ rumen degradability study

Statistical analysis

Results and Discussion

Conclusions

Acknowledgements

References

Effect of different particle lengths on the bacterial population, fermentation profiles and nutritive value of whole maize plant silage

K T Khaing, T C Loh2, S Ghizan1, R A Halim1 and A A Samsudin2

Abstract

Introduction

Methods

Silage preparation and sampling

Microbial analysis

pH and fermentation analysis

Chemical analysis

In situ rumen degradability study

Statistical analysis

Results and Discussion

Conclusions

Acknowledgements

References

K T Khaing, T C Loh², S Ghizan¹, R A Halim¹ and A A Samsudin²