Nutritional status of corn silage after probiotic conservation

Abstract

The effects of two probiotic inoculants Enterococcus faecium 2/3s (EF2/3s) and E. faecium 26/42 (EF26/42) on nutritional patterns and fatty acid concentration in corn silages were examined. Both inoculants were isolated and characterized at Institute of Animal Physiology Slovak Academy of Sciences. The whole crop corn (341 g of dry matter (DM) kg⁻¹) was conserved at 21°C for 111 days during the three treatments: (1) corn without inoculant (CS, control), (2) corn inoculated by the strain EF2/3s (CS + EF2/3s) and (3) corn inoculated by the strain EF26/42 (CS + EF26/42). Counts of the inoculated strains were enumerated (5.58 and 4.92 log₁₀ cfu g⁻¹ for EF26/42 and EF2/3s, respectively) immediately after their inoculation. At the end of conservation, EF26/42 achieved 1.60 ± 0.26 log₁₀ cfu g⁻¹ and EF2/3s less than 1.0 log₁₀ cfu g⁻¹. Compared to CS, the lactate values of inoculated CS were greater (P < 0.001). It can be concluded that both bacterial inoculants effectively enhanced in vitro DM degradability and lactic acid concentration in corn silages, but the effect on fatty acids concentration was inconsistent.

Keywords:

• corn silage
• fatty acids
• fermentation biotechnology
• nutritive value

1. Introduction

Whole crop corn is a popular silage crop for feeding ruminants because it is easily digestible and highly nutritious (Li & Nishino 2011). The effects of bacterial inoculants on silage fermentation accompanied by reduced pH, promoted lactic acid production and reduced ammonia formation when compared with uninoculated silage (Muck & Kung 1997). Among lactic acid bacteria (LAB), the most commonly used are Lactobacillus plantarum, Pediococcus acidilactici and/or Enterococcus faecium. The in vitro screening of micro-organisms at our Institute of Animal Physiology (Marcináková 2006) showed that some lactobacilli and enterococci isolated from rumen fluid and silages were able to convert linoleic acid to conjugated linoleic acids (cis9 trans11 C18:2). Some of the strains from this screening were successfully used as inoculants for the conservation of corn (Jalč et al. 2009a) or grass (Jalč et al. 2009b). Numerous papers published the ensiling of corn without inoculants or with the inoculants as L. plantarum, Lactobacillus buchneri, Propionibacterium acidipropionici or commercial inoculants containing lactobacilli, enterococci, pediococci and with the mixtures of the inoculants, respectively (Filya 2003; Weinberg et al. 2004; Sucu & Filya 2006; Filya & Sucu 2007; Koc et al. 2008; Huisden et al. 2009). The probiotic inoculants (E. faecium EF2/3s and EF26/42) have the ability to sufficiently establish in grass silages and consequently improve the quality of silage (Váradyová et al. 2013). Then, the objectives of the present experiment were (1) to find out the ability of inoculants (E. faecium EF2/3s and EF26/42) to survive in whole crop corn silage, and (2) to study the effect of these inoculants on microbial parameters, nutrient composition and fatty acid concentration in corn silage after 111 days of conservation.

2. Materials and methods

2.1. Experimental design

The silages were made from whole crop corn. The agronomic details for the corn were as follows: sowing date – 22 April 2011; harvesting date – 15 September 2011; variety – Pioneer 37M34 (FAO 430); irrigation – once pre-sowing and six times after sowing; and fertilizer applications − 300 kg ha⁻¹ Duslofert (Duslo Šala, Slovakia) containing N:P:K (15:15:1), where N is in the form of ammonium nitrate, P is P₂O₅ and K is K₂O. About 50 kg of the whole crop corn, after wilting for 16 h, was chopped to a length of 20 mm with a forage chopper. This amount was then divided into three parts, and the conservation of the corn was carried out in 36 polyethylene jars. Three silage treatments (12 jars in each) were used: (1) corn without inoculant (CS, control), (2) corn inoculated with the strain E. faecium EF2/3s (CS + EF2/3s) and (3) corn inoculated with the strain E. faecium EF26/42 (CS + EF26/42). The strains were cultivated in MRS broth (Lactosebacillus Broth acc. to DE MAN, ROGOSA and SHARPE, Merck, Darmstadt, Germany) at 37°C for 18 h. The cultures were centrifuged (10,000× g) for 30 minutes, and the cells were re-suspended in a quarter-strength Ringer solution (pH 7.0, Oxoid, Basingstoke, England) to reach a count of 10⁹ cfu mL⁻¹ for each. The cultures were applied in a concentration of 10⁹ cfu mL⁻¹ in Ringer solution (10 mL kg⁻¹ of fresh whole corn plants). The control corn was sprayed with 10 mL of distilled water per kg of corn. The whole crop corn for all treatments was hand pressed (900–1000 g) into 1-L jars. The conservation of corn in the jars was carried out at an average temperature of 21°C (the temperature was recorded daily) for 111 days, and the jars were sealed with a lid and joints filled with paraffin to prevent the entry of oxygen. Neither the jars nor their lids allowed light to enter. Effluent and gas were not discarded from the jars during conservation. Representative samples were collected from five different sites of the 50 kg whole crop corn, and average samples (in triplicate) were used for microbiological and chemical analyses before the division into jars (day 0–1). In addition, two jars per treatment were opened on days 7, 40 and 111 of conservation for microbiological analyses (in triplicate, total six samples), and another two jars per treatment were opened for nutritional analyses and for fermentation parameters (in triplicate, total six samples) after conservation.

2.2. Chemical analysis

After conservation, two jars per treatment were used for nutritional analyses and determination of the fermentation patterns of the corn silage extracts. Samples from two jars were collected from five different locations, and the average samples were analysed. Chemical analysis is presented on a dry matter (DM) basis. Samples of whole crop corn and corn silages were analysed for neutral detergent fibre (NDF), acid detergent fibre (ADF; Van Soest et al. 1991) and acid detergent lignin (ADL) using Fibertec 2010 (Tecator Comp., Höganäs, Sweden). In vitro DM digestibility (IVDMD) of the whole crop corn and corn silages was determined from the difference between the substrate weight before and after the 72-h incubation of batch cultures with rumen inocula (Mellenberger et al. 1970). Standard methods (Association of Official Analytical Chemists 1990) were used to determine the DM (No. 967.03), ash (No. 942.05), nitrogen (No. 968.06), fat (No. 983.23) and crude protein (No. 990.03).

Silage water extracts were measured for pH (pH metre, Inolab, level 1, Weilheim, Germany), lactic acid, short-chain fatty acids (SCFAs) and ammonia nitrogen (Association of Official Analytical Chemists 1990). Fatty acids (FA) composition (g kg⁻¹ of FA) in fresh corn and corn silages were determined in lyophilised samples. Lipids were extracted from 500 mg of freeze-dried sample with a mixture of chloroform: methanol (2:1) according to Bligh and Dyer (1959). The FA methyl esters peaks were identified by authentic standards of C4–C24 FA methyl ester mixture (Supelco, Bellefonte, PA, USA).

2.3. Statistical analysis

Statistical analysis used analysis of variance (Graphpad Instat, Graphpad Software Inc., San Diego, CA, USA) as a repeated measures model that represented four groups (i.e., crop corn, corn silage without inoculants, corn silage with EF2/3s and corn silage with EF26/42). When the overall treatment effect was significant (P < 0.05), individual treatment differences were determined using Tukey's post-test.

3. Results and discussion

Sufficient counts of both inoculated strains EF2/3s and EF26/42 were enumerated (5.58 and 4.92 log₁₀ cfu g⁻¹ for EF26/42 and EF2/3s, respectively) immediately after their inoculation (Table 1). Although during the conservation process they decreased (not significantly), at the end of conservation, EF26/42 reached 1.60 ± 0.26 log₁₀ cfu g⁻¹ and fewer than 1.0 log₁₀ cfu g⁻¹ colonies of EF2/3s were counted. The balanced counts of the total LAB, enterococci, amylolytic streptococci and coliform bacteria were detected in the pre-ensiled materials. Although the counts of the total LAB were gradually increased (not significantly) till 40 days of ensiling, their counts in the silage were diminished at the end of conservation. The counts of the total enterococci, amylolytic streptococci and coliform bacteria were decreased (not significantly) during the conservation process (taking into account the start and end of conservation). Coliform bacteria had already decreased (not significantly) on Day 7 of conservation. The numerical decrease in the microbiota could be explained by the cross-competition of LAB or enterococci with the other microbiota and/or it could be due to the bacteriocin activity of the inoculant strain EF26/42, when bacteriocins from LAB may also have inhibitory activity against other LAB (Settanni et al. 2005). The results of microbiological testing corroborated the statement that the natural population of LAB in pre-ensiled corn is often as high as that of the inoculant (approximately 10⁶ log₁₀ cfu g⁻¹); it, therefore, may be difficult to exceed the indigenous LAB by inoculating corn silage (Hu et al. 2009; Kristensen et al. 2010). Both inoculated strains survived in the corn silage, although their counts were numerically decreased at the end of conservation when enterococci are less pH tolerant as many other LAB. The enterococci and streptococci were able to survive at pH 4.1 in corn silage during conservation of three weeks (Peterson-Wolfe et al. 2011).

Table 1. The counts of inoculants (log₁₀ cfu g⁻¹) in corn silages (CS) during ensiling.

Silages	Inoculants	LAB	Enterococci	Amylolytic streptococci	Coliforms bacteria
Day 0–1
CS	–	5.60 ± 2.36	5.08 ± 2.25	5.29 ± 2.30	5.32 ± 2.30
CS+EF2/3s	4.92 ± 2.21	5.88 ± 2.42	5.37 ± 2.31	5.50 ± 2.34	4.46 ± 2.11
CS+EF26/42	5.58 ± 2.36	5.73 ± 2.39	5.99 ± 2.44	6.76 ± 2.42	4.50 ± 2.12
Day 7
CS	–	6.43 ± 2.49	6.31 ± 2.51	4.31 ± 2.07	2.39 ± 1.54
CS+EF2/3s	3.71 ± 1.92	7.30 ± 2.70	5.30 ± 2.30	6.29 ± 2.50	2.26 ± 1.50
CS+EF26/42	3.24 ± 0.80	7.36 ± 2.71	6.71 ± 2.59	6.59 ± 2.56	2.67 ± 1.63
Day 40
CS	–	6.95 ± 2.63	4.04 ± 2.00	3.10 ± 1.76	<1.0
CS+EF2/3s	2.66 ± 1.62	6.71 ± 2.59	4.04 ± 2.00	3.57 ± 1.88	<1.0
CS+EF26/42	2.74 ± 1.65	4.56 ± 0.14	2.77 ± 0.66	3.79 ± 1.94	<1.0
Day 111
CS	–	4.62 ± 0.14	2.41 ± 0.55	1.99 ± 0.41	<1.0
CS+EF2/3s	<1.0	4.83 ± 0.19	3.22 ± 0.79	2.36 ± 0.53	1.42 ± 0.19
CS+EF26/42	1.60 ± 0.26	4.56 ± 0.14	2.77 ± 0.66	4.52 ± 0.12	2.58 ± 0.60

Values are means ± SD (standard deviation).

The nutrient composition of whole crop corn (C) and corn silages (i.e., CS, CS+EF2/3s and CS+EF26/42) as well as fermentation parameters of corn silages are shown in Table 2. The conservation process led to a numerical decrease in the DM concentration in all of the corn silages with respect to the mean DM content in whole crop corn (341 g kg⁻¹). IVDMD was CS + EF2/3s = CS + EF26/42 > CS > C (687, 684, 617 and 514 g kg⁻¹ of DM, respectively). Similar results were shown when the mixture of inoculants (L. plantarum + Pediococcus acidilactici) and enzyme (amylase) with a concentration of 5 × 10⁵ and 1 × 10⁶ log₁₀ cfu g⁻¹ in corn silage was used (Koc et al. 2008). It is known that DM losses during conservation are a useful indicator of L. buchneri activity in silages inoculated with this bacterium (Driehuis et al. 2001). When the forage was treated with homofermentative LAB (Pediococcus pentosaceus and L. plantarum), lower DM losses were also reported (Driehuis et al. 2001). However, inoculation of corn silage with L. buchneri and L. plantarum or a mixture of the two did not affect in situ DM and organic matter degradability after 48 h of fermentation (Filya 2003). Our results revealed that value of DM in crop corn was higher (P < 0.05), whereas the IVDMD of crop corn incubated in rumen fluid was lower (P < 0.001) compared with the corn silages. This was caused by the higher NDF and ADL (P < 0.001) in the crop corn. In CS + EF26/42, the decline of NDF and ADF was probably caused by slight hydrolysis of the cell wall. In addition, some authors (Baytok et al. 2005; Koc et al. 2008) reported a significant decrease in NDF and ADF contents in corn silages after inoculation with L. plantarum + P. acidilactici inoculants. In our experiment, the mean pH values were decreased (P < 0.001) during 111 days conservation from 5.3 (C) to 3.77 (CS+EF26/42), 3.78 (CS+EF2/3s) and 3.79 (CS). Studies have shown that the success rate of LAB inoculation is less in corn silage than in grass silage (Muck & Kung 1997) because bacterial additives are less frequently used in corn silage. Compared with CS, the lactate values and the lactate-to-acetate ratio of inoculated CS were greater (P = 0.001) mainly in CS + EF26/42. In general, lactic acid should be at least 65–70% of the total silage acids in a well-fermented silage. Lactic acid concentration in CS, CS + EF2/3s and CS + EF26/42 represented 57.5%, 62.1% and 79.0%, respectively, of the total silage acids. Regarding the fermentation profile, more lactic acid was found in CS + EF26/42, which was consistent with the tendency towards the lower pH value (Table 2). The concentrations of acetic acid in corn silages ranged from 1.89% to 2.09%, but the concentrations of propionic and n-butyric acids were not detectable. Previously, average concentrations of acetic (1–3%), propionic (<0.1%) and n-butyric acids (0%) in corn silages (DM, 30–40%) were reported (Kung & Shaver 2001). Our results confirm the hypothesis (Filya et al. 2000) that the homofermentative LAB used mainly produced not only lactic acid but also small amounts of SCFAs (acetate, propionate or n-butyrate). The ratio of lactic acid to acetic acid is a good indicator of the efficiency of silage fermentation (Kung et al. 2000), and ideally, this ratio should not be less than 3:1 (Kung & Shaver 2001). The addition of inoculant enterococci increased the lactic acid to acetic acid ratio from 1.35:1 (CS) to 1.62:1 (CS + EF2/3s) or 3.77:1 (CS + EF26/42). Our results showed that the total silage acid concentration, which indicates the extent of fermentation during conservation, was 1.2 times higher in CS + EF2/3s, and 2 times higher in CS + EF26/42 than in CS. Ammonia concentration can be expressed as a percentage of the crude protein (CP), and ideally, it should be less than 0.9% of the CP content. Our results showed that the proportion of ammonia nitrogen in CP (g kg⁻¹ DM) content is 0.6%, 0.7% and 0.7% for the CS and CS + EF2/3s and CS + EF26/42, respectively.

Table 2. Chemical composition in whole crop corn and corn silages after 111 days of conservation.

		Corn silage
	Crop corn	–	EF2/3s	EF26/42	SEM	P-value
DM (g kg^−1)	341^Footnoteb	320^Footnotea	314^Footnotea	318^Footnotea	8.4	*
Nitrogen (g kg^−1 DM)	10.7	11.7	11.7	11.4	0.86	Ns
Crude protein (g kg^−1 DM)	66.7^Footnotea	73.2^Footnoteb	73.1^Footnoteb	71.2^Footnoteb	1.29	**
NDF (g kg^−1 DM)	448^Footnotec	410^Footnoteb	417^Footnoteb	388^Footnotea	3.39	***
ADF (g kg^−1 DM)	243^Footnoteb	245^Footnoteb	245^Footnoteb	219^Footnotea	2.08	***
ADL (g kg^−1 DM)	44.5^Footnotec	24.9^Footnotea	31.6^Footnoteb	27.1^Footnotea	1.33	***
Fat (g kg^−1 DM)	22.8	30.0	24.7	29.2	1.08	Ns
Ash (g kg^−1 DM)	49.1^Footnoteb	52.1^Footnoteb	59.1^Footnotec	45.4^Footnotea	1.05	***
IVDMD (g kg^−1 DM)	514^Footnotea	617^Footnoteb	687^Footnotec	684^Footnotec	12.1	***
pH	5.30^Footnoteb	3.79^Footnotea	3.78^Footnotea	3.77^Footnotea	0.04	***
Lactate (g kg^−1 DM)	–	26.2^Footnotea	33.8^Footnoteb	71.0^Footnotec	0.91	***
Acetate (g kg^−1 DM)	–	19.4	20.9	18.9	1.04	Ns
Lactate: acetate	–	1.35^Footnotea	1.62^Footnoteb	3.77^Footnotec	0.03	***
NH3N (mg N 100 g^−1 DM)	–	43.0^Footnotea	48.2^Footnoteb	52.5^Footnotec	0.81	***

^a–cMeans within a row with no common superscript differ significantly (P < 0.05).

In general, not only the alterations in FA composition of corn may be due to microbial intervention during the process of conservation but also the enzymes of herbal origin might be active during conservation (Jalč et al. 2009a). The FA composition of whole crop corn before conservation and corn silages after 111 days of conservation is shown in Table 3. Compared with the CS and inoculated CS, the C showed a lower (P < 0.01) concentration of cis C18:2n-6 linoleic and a higher (P < 0.01) concentration of α-linolenic acid. The content of γ-linolenic acid in CS + EF2/3s was higher (P < 0.01) when compared with that of C, CS and CS + EF26/42. The concentrations of saturated FA, unsaturated FA, medium-chain FA and long-chain FA did not differ across treatments. The effect of conservation on total FA content of forages is inconsistent, with some studies reporting their decrease (Elgersma et al. 2003) and/or an increase (Boufaied et al. 2003). In our experiment, changes in the proportions of C18:2n-6, C18:3n-3 and C18:3n-6 were recorded. Decreased proportions of C18:2n-6 and C18:3n-3 in corn silages inoculated with commercial bacteria L. plantarum, E. faecium, P. acidilactici and Lactobacillus salivarius compared with fresh corn have been reported previously (Alves et al. 2011).

Table 3. Concentration of fatty acids (g kg⁻¹ of FA) in whole crop corn and corn silages after 111 days of conservation.

		Corn silage
	Crop corn	–	EF2/3s	EF26/42	SEM	P-value
C8:0 caprylic	0.89^Footnotec	2.03^Footnoted	0.66^Footnoteb	0.39^Footnotea	0.124	***
C10:0 caprinic	5.07^Footnoted	4.13^Footnotec	2.30^Footnoteb	1.21^Footnotea	0.411	***
C12:0 lauric	18.3^Footnotec	13.0^Footnoteb	11.0^Footnoteb	7.98^Footnotea	0.962	***
C14:0 myristic	11.9^Footnotec	8.20^Footnoteb	9.51^Footnoteb	6.97^Footnotea	0.991	***
C16:0 palmitic	217^Footnotea	238^Footnotec	236^Footnotec	225^Footnoteb	12.4	***
C18:0 stearic	38.8^Footnoteb	29.5^Footnotea	30.7^Footnotea	32.3^Footnotea	2.97	***
Cis C18:1n-9 oleic	187	193	180	196	7.3	Ns
Cis C18:2n-6 linoleic	300^Footnotea	366^Footnoteb	348^Footnoteb	374^Footnoteb	8.1	**
C18:3n-3 α-linolenic	180^Footnoteb	122^Footnotea	144^Footnotea	120^Footnotea	6.5	**
C18:3n-6 γ-linolenic	0.29^Footnotea	0.06^Footnotea	0.82^Footnotec	0.47^Footnoteb	0.001	^Footnote***
Total C18 FA	707	711	706	724	18.1	Ns
Saturated FA	313	308	307	289	10.2	Ns
Unsaturated FA	686	693	694	710	16.8	Ns
Short-chain FA	26.0^Footnotec	20.0^Footnoteb	14.5^Footnotea	10.3^Footnotea	0.92	***
Medium-chain FA	240	251	259	244	7.3	Ns
Long-chain FA	734	728	724	745	15.7	Ns

^a–dMeans within a row with no common superscript differ significantly (P < 0.05).

4. Conclusion

It can be concluded that both of the bacterial inoculants (i.e., E. faecium 2/3s and E. faecium 26/42) isolated in our laboratory survived in the corn silage, although their counts were numerically decreased at the end of conservation when enterococci were less pH tolerant. Both inoculants (E. faecium 2/3s and E. faecium 26/42) effectively enhanced IVDMD and lactic acid concentration in corn silages, but the effect on FA concentration was inconsistent. We should like to use the results from our treatments for in vivo studies; final results could be obtained in agricultural practice.

Additional information

Funding

The study was supported by the project VEGA [grant number No. 2/0009/14] of the Grant Agency of the Ministry of Education of Slovak Republic and Slovak Academy of Sciences.