S H O R T R E P O R T
Effects of wilting and lactic acid bacteria inoculation on fermentation and microbial community of elephant grass silage produced in Vietnam
Thi Minh Tu Tran1,2 , Mui Thi Nguyen3, Huu Van Nguyen3and Naoki Nishino1
1 Graduate School of Environmental and Life Science, Okayama University, Okayama, Japan
2 Faculty of Agriculture, Engineering and Food Technology, Tien Giang University, My Tho City, Vietnam 3 Faculty of Animal Science, Hue University of Agriculture and Forestry, Hue City, Vietnam
Keywords
Microbiota; silage; tropical grass.
Correspondence
Naoki Nishino, Graduate School of Environ- mental and Life Science, Okayama University, Kita-ku, Okayama 700-8530, Japan.
Email: [email protected] Received 28 June 2017;
accepted 28 August 2017.
doi: 10.1111/grs.12187
Abstract
Direct-cut (dry matter [DM] 165 g kg1) and wilted (DM 250 g kg1) ele- phant grass silages were prepared in Hue, Vietnam, with and without a mixture ofLactobacillus paracasei and Lactococcus lactis (LP+LC) or Lactobacillus buch- neri (LB). The silages were stored for 4 months, and fermentation products and aerobic stability were determined. The microbial community was assessed using denaturing gradient gel electrophoresis and quantitative polymerase chain reaction. Acetate was predominantly produced during the fermentation of con- trol and LB-inoculated silages, whereas the content was not sufficient to inhibit aerobic spoilage for 7 days in control silage. The LP+LC inoculation greatly enhanced lactate content, suppressed the alcohol content, and did not improve aerobic stability or decrease the total fungal population. The bacterial commu- nity and L. buchneri populations were similar between control and LB-inocu- lated silages; however, Kluyveromyces delphensiswas undetectable and the total fungal population was lowered in LB-inoculated silage.
Introduction
In tropical environments, biomass production is rapid and enormous, and thus forage supply for ruminant livestock can be stabilized if proper preservation is undertaken (Van 2012). Ensiling has become a valuable process though it is still a difficult task to obtain accept- able storability and stability (Nussio 2005). Tropical grass ensiling has often been demonstrated to produce acetic acid or alcohol as the major fermentation product (Nishino et al. 2012), and the high ambient tempera- tures may deteriorate stability after opening the silo (Ashbell et al. 2002). Even if lactic acid dominates the fermentation in the initial ensiling period, the acetic acid content may increase and lactic acid content may decrease when ensiling is prolonged. Unlike temperate grass ensiling, wilting and lactic acid bacteria (LAB) inoculation cannot ensure lactic acid fermentation (Umana et al. 1991). Molasses addition is not firmly effective either, indicating that sugar deficiency in the
pre-ensiled crop may not be a critical factor for acetic acid fermentation (Nishino et al.2012).
Sufficient data on the microbial community involved in tropical grass ensiling are as yet unavailable; hence, we have conducted a number of culture-independent micro- bial analyses on guinea grass silage, a typical tropical for- age used in the south-west of Japan. Parvin and Nishino (2009, 2010) reported thatLactobacillus plantarum, Lacto- bacillus brevis, Lactococcus lactis and Pediococcus pen- tosaceus were associated with ensiling, and Nishino et al.
(2012) demonstrated that enterobacteria, such as Pantoea sp. and Morganella sp., could be involved in the acetic acid fermentation of silage. Although these findings helped to improve our knowledge and understanding about the types of microorganisms that can be engaged in tropical grass ensiling, results were not straightforward and further studies are still necessary to clarify microbiota involving acetic acid fermentation.
In this study, elephant grass (Pennisetum purpureum) silage was produced in Hue, Vietnam, and its microbiota
Japanese Society of Grassland Science ISSN1744-6961
was examined after transfer to Okayama, Japan, which enabled the analysis of tropical grass silage produced under its exact indigenous environment. Commercial homo-fermentative and hetero-fermentative LAB inocu- lants were tested to investigate how fermentation and microbiota could be altered and influenced in tropical regions. Furthermore, both direct-cut and wilted silages were prepared, because fermentation patterns and efficacy of LAB inoculants can be differentiated by wilting prior to ensiling. The objective was to clarify the microbial association with elephant grass ensiling and evaluate homo-fermentative and hetero-fermentative LAB inocu- lants in regard to fermentation control and aerobic stabil- ity management in tropical environments.
Materials and methods
Elephant grass (P. purpureum cv. VA06) was manually harvested at the early ear emergence stage with a plant height of 100–120 cm on 5 July 2014 in Hue, Vietnam (16°300N, 107°350E, Vietnam). The grass was cut 15–
20 cm above ground level, wilted for 6 h with occasional turning, and then shredded, using knives, into 10–20- mm pieces before ensiling. This cultivar grows up to 300–500 cm when it matures, but Vietnamese farmers practice frequent cutting, with an interval of 30–45 days.
Half of the shredded grass was ensiled without wilting.
For inoculants, a mixture of L. paracasei and L. lactis (LP+LC; Simaster LP, Snow Brand Seed Co. Ltd., Sap- poro, Japan) and L. buchneri (LB; Silo SP, Snow Brand Seed Co. Ltd.) were dissolved in deionized water and then sprayed onto the pre-ensiled material at 19108cfu g1. Silage with the same amount of water sprayed served as a control. In total, 300 g of shredded grass was packed in a plastic pouch (Hiryu BN-12; Asahi Kasei Pax, Tokyo, Japan), and the air was removed using a vacuum sealer. The size, thickness, and oxygen perme- ability of the pouch were 2709 400 mm, 0.075 mm and 44 mL m2 atm1 day1, respectively. Triplicate silos were stored in Hue, Vietnam, for 4 months at room temperature and then transferred to Okayama, Japan, as check-in aircraft luggage. For this transportation, approvals from animal and plant quarantine authorities were obtained.
The dry matter (DM) contents of pre-ensiled crops and silages were determined after drying the mass in an oven at 60°C for 48 h. The pH value and lactic acid, volatile fatty acids, and alcohol content were determined in cold water extracts (Tranet al.2014). Aerobic spoilage test was carried out as follows. After the silage was opened, 100 g of the contents were put into polyethylene bottles (500 mL) without compaction. The top of the bottle was kept uncovered and exposed to air at 30°C for
7 days. The pH value was determined to ascertain spoilage.
Microbial communities were determined using dena- turing gradient gel electrophoresis (DGGE). Bacterial and fungal DNA extraction, primer sets and conditions for polymerase chain reaction (PCR) amplification, and clon- ing and sequencing of DGGE bands were performed according to the procedures described by Li and Nishino (2011) and Ni et al. (2017). Searches in the GenBank database with the BLAST program were performed to determine the closest relatives of the 16S rRNA (bacteria) and 18S rRNA (fungi) gene sequences. Positive identifica- tion of unknown sequences was made when more than 99% identity was obtained in BLAST analysis.
Quantitative PCR (qPCR) was carried out on a MiniOpticon System (Bio-Rad Laboratories, Inc., Tokyo, Japan). Primers for total fungi (forward: 50-GCAAGTC TGGTGCCAGCAGCC-30; reverse: 50-TTGGCAAATGC TTTCGC-30), total bacteria (forward: 50-CCTACGGGAG GCAGCAG-30; reverse: 50-ATTACCGCGGCTGCTGG-30), Lactobacillus spp. (forward: 50-AGCAGTAGGGAATCTT CCA-30; reverse: 50-CACCGCTACACATGGAG-30), and L. buchneri (forward: 50-GAAACAGGTGCTAATACCGTA TAACAACCA-30; reverse: 50-CGCCTTGGTAGGCCGTTAC CTTACCAACA-30) were used for quantification. The PCR cycle parameters for the total fungi assay were as follows:
5 min at 95°C and 35 cycles of 15 s at 95°C, 20 s at 50°C, and 30 s at 72°C. The annealing temperatures for total bacteria, Lactobacillus spp., and L. buchneri assays were 60°C, 58°C, and 61°C, respectively. Serial dilution series of plasmids carrying the nearly full-length 18S rRNA gene ofSaccharomyces cerevisiae(for total fungi) and 16S rRNA gene of Escherichia coli (for total bacteria), L. plantarum (for Lactobacillus spp.), and L. buchneri were used as the known standard concentrations.
Data were subjected to two-way analysis of variance, with wilting and LAB inoculation as factors. When a sig- nificant difference was found due to LAB inoculation, the effects were compared using Tukey’s multiple compari- son. Differences were considered significant when the probability was < 0.05. These analyses were carried out using JMP software (ver. 11; SAS Institute, Tokyo, Japan).
Results and discussion
The contents of DM and water soluble carbohydrates (WSC) were 165 g kg1 and 40 g kg1DM for direct-cut material and 250 g kg1 and 35 g kg1 DM for wilted material, respectively. In direct-cut control silage, acetic acid was found as the main fermentation product, fol- lowed by lactic acid and ethanol being produced at levels comparable to each other (Table 1). A small amount of
1,2-propanediol was also observed in control silage. Inoc- ulation of LP+LC greatly enhanced the lactic acid content and decreased the content of acetic acid, ethanol, and 1,2-propanediol. The amount of acids and alcohols (110 g kg1DM) determined in LP+LC-inoculated silage far exceeded the content of WSC in the pre-ensiled crop (40 g kg1DM); hence, components other than WSC, for example, pectin and starchy substances, may have been metabolized by the inoculated LAB species. Inoculation of LB increased the acetic acid content and decreased the ethanol content. AlthoughL. buchneriis known to metab- olize lactic acid to acetic acid and 1,2-propanediol (Oude Elferink et al. 2001), the changes in the lactic acid and 1,2-propanediol contents were not distinctive in this study. Nevertheless, aerobic spoilage was efficiently sup- pressed by LB treatment; the pH value remained as low as 4.62 even when the silage was exposed to air at 30°C for 7 days. Wilting increased the ethanol and 1,2-propa- nediol contents, without affecting the lactic acid and acetic acid content. The effects of LAB inoculation appeared to be the same as those seen in direct-cut silage;
LP+LC intensified the lactic acid fermentation and LB fortified the acetic acid fermentation. Inhibition of aero- bic spoilage was also observed in LB-inoculated wilted silage. The effect of wilting was restricted to suppressing alcoholic fermentation in this study, whereas similar moisture reduction was sufficient to change both acid and alcohol contents in temperate grass silages (Nishino and Touno 2005).
As acetic acid was the main fermentation product in control silage, good aerobic stability could be observed even without LB inoculation. However, if the acetic acid content was expressed as un-dissociated forms, the aver- age values for direct-cut and wilted control silage were 3.22 and 3.25 g kg1 fresh matter (FM) and those for direct-cut and wilted LB-inoculated silage were 5.84 and 9.18 g kg1 FM, respectively. Wilkinson and Davies (2012) indicated that silages with un-dissociated acetic acid content lower than 3 g kg1 FM were unstable in air, and 8 g un-dissociated acetic acid kg1 FM was nec- essary to ensure a low risk of aerobic instability. The con- centrations of control silages were close to the critical level, whereas those of wilted LB-inoculated silage were greater than the stable level. In fact, little pH change was observed during the 7-day aerobic stability test in wilted LB-inoculated silage.
Bands of L. brevis (band 1), L. buchneri (bands 4 and 8), andL. paracasei (band 7) were found in control silage regardless of the wilting, and almost the same band pro- file was seen in LB-inoculated silage (Figure 1). Inocula- tion of LP+LC altered the bacterial community greatly;
L. buchneri and L. brevis appeared to be eliminated, whereas L. paracasei remained detectable in the silage.
AlthoughL. lactis was included in the LAB inoculant, the band of L. lactis (band 3) was not seen in LP+LC-inocu- lated direct-cut silage. The band of L. lactis was distinc- tive in the control and LB-inoculated silages irrespective of wilting, and also in LP+LC-inoculated silage with
Table 1 Fermentation characteristics and microbial populations of direct-cut and wilted elephant grass silages prepared with and without homo- fermentative and hetero-fermentative lactic acid bacteria inoculants and stored for 4 months in Hue, Vietnam
Direct-cut silage Wilted silage
SE
ANOVA
Control LP+LC LB Control LP+LC LB W LAB W9LAB
Fermentation characteristics
DM (g kg1) 156b 189a 159b 248y 261x 245y 12.10 ** ** NS
pH 4.20a 3.40b 4.28a 4.42x 3.43y 4.24x 0.07 NS ** NS
Lactic acid (g kg1DM) 15.80b 96.80a 2.21b 13.30y 105x 13.10y 7.03 NS ** NS
Acetic acid (g kg1DM) 26.30b 2.45c 48.90a 19.10y 3.79z 48.80x 2.95 NS ** NS
Ethanol (g kg1DM) 17.70a 10.10c 13.00b 22.00x 11.60z 17.90y 1.33 ** ** NS
1,2-Propanediol (g kg1DM) 3.17a 0.58b 2.97a 12.00x 2.03y 11.70x 1.24 ** ** * pH after 7 days spoilage test 6.05a 6.56a 4.62b 5.73x 7.23x 4.36y 0.46 NS ** NS Microbial populations
Total bacteria (log10 copies g1) 9.65a 7.39b 9.68a 10.10x 7.24y 9.95x 0.09 * ** *
Lactobacillus(log10 copies g1) 9.07a 6.22b 8.88a 9.60x 6.15y 9.22x 0.16 NS ** NS
L. buchneri(log10 copies g1) 8.62a 5.42b 8.63a 8.58x 5.75y 8.57x 0.22 NS ** NS
Total fungi (log10 copies g1) 7.97b 8.66a 7.83c 8.18y 8.55x 7.79z 0.09 NS ** NS Mean values for triplicate silages. Values in the same row with different following letters (a–c, x–z) are significantly different (P<0.05). ANOVA, analysis of variance; DM, dry matter; LAB, lactic acid bacteria inoculant; LB,Lactobacillus buchneri; LP+LC,Lactobacillus paracasei+Lactococcus lactis; W, wilting.
*P< 0.05; **P< 0.01; NS, not significant.
wilting prior to ensiling. Several bands indicative of uncultured bacteria (bands 9 and 10) appeared in the LP+LC-inoculated silages newly. The finding that most bands were shared between the control and LB-inoculated silage was confirmed in both direct-cut and wilted silages.
The populations of total bacteria (approximately 9.6 log copies g1), Lactobacillus spp. (approximately 9.0 log copies g1), andL. buchneri(approximately 8.6 log copies g1) were similar between control and LB-inoculated silage. Decreases of about 103fold were seen in LP+LC- inoculated silage. The bacterial population may have declined faster after attaining stationary growth due to intensified fermentation. The bacterial community analy- sis, which showed elimination ofL. brevisandL. buchneri by LP+LC treatment, could account for how hetero-fer- mentative LAB activities were involved in acetic acid fer- mentation of elephant grass silage.
The fungal community was greatly modified by the LP+LC and LB treatments (Figure 1). Although Wicker- hamomyces anomalus (band 11), Kluyveromyces delphensis (band 12),Candida glabrata (band 14), andPichia kudri- avzevii (band 16) were observed in the control silage regardless of wilting, bands of C. glabrata and P. kudri- avzevii were absent in the LP+LC- and LB-inoculated silages. Further, bands of K. delphensis(bands 12 and 13) were faint in LB-inoculated silage. Candida glabrata was isolated from the effluent of grass silage (Olsen and Ped- ersen 1974) and was detectable in DGGE analysis of spoiled corn fodder (Li and Nishino 2011). Likewise,
P. kudriavzevii was isolated from corn silage exposed to air (Middelhoven and van Baalen 1988). However, despite the fact that C. glabrata is regarded as a yeast species involved in aerobic spoilage, the association of P. kudri- avzeviihas not been clearly demonstrated. Tsegaye (2016) reported thatP. kudriavzeviiand K. delphensiswere toler- ant to temperatures up to 37°C and grew well in 160 and 60 mL L1of ethanol. High storage temperatures in Viet- nam, therefore, may have facilitated the growth of P. ku- driavzeviiand K. delphensis.
No unusual bacteria and fungi were found in the con- trol silage with and without wilting. Both L. brevis and L. buchneri are known as representative hetero-fermenta- tive LAB species in silage (Muck 2013), but the metabolic activity ofL. buchneri with respect to lactic acid degrada- tion (Oude Elferink et al. 2001) may account for the greater acetic acid content than the lactic acid content in control silage. A small amount of 1,2-propanediol and a large population of L. buchneri (determined by qPCR) could support this. However, despite the occurrence of typical acetic acid fermentation, our previous studies did not identify L. buchneri in rhodes grass or guinea grass silages (Parvin and Nishino 2009, 2010); hence, the involvement of L. buchneri in acetic acid fermentation would not consistently occur in tropical grass ensiling.
Even so, the activity of indigenous L. buchneri was not sufficient to secure stability after exposure to air. The use of commercial LB inoculant further encouraged acetic acid fermentation, which inhibited W. anomalus, K. delphensis, C. glabrata, and P. kudriavzevii, and decreased the total fungal population.
The LP+LC inoculant was excellent in regard to enhancing lactic acid fermentation, because the benefit was sustained for 4 months under the exact tropical envi- ronment. Unlike in temperate crops, it has been shown to be difficult to ensure improved fermentation by homo- fermentative LAB inoculation in tropical grass ensiling (Umana et al. 1991). Moreover, suppression of alcohol production was greater than that in LB-inoculation, sug- gesting that LP+LC may have well competed with yeasts in the initial ensiling period. Indeed, C. glabrata and P. kudriavzevii were undetectable upon silo opening.
However, K. delphensis remained as a dense band in the DGGE analysis and the total fungal population was increased in LP+LC-inoculated silage. Because aerobic sta- bility was not improved by LP+LC treatment,K. delphen- sis was regarded as the most important yeast to inhibit the aerobic spoilage of elephant grass silage.
In conclusion, hetero-fermentative L. brevis and L. buchneri may be involved in acetic acid fermentation of elephant grass silage. Wilting may not be effective in enhancing desirable fermentation. Although acetic acid fermentation can be avoided by the use of selected homo-
Bacteria
Control LP + LC LB
Direct-cut
Control LP + LC LB
Wilted
Fungi
Control LP + LC LB
Direct-cut
Control LP + LC LB
Wilted 1 2
4 3 5
6 7 9 10
12 11
15 14 16 17
Lactobacillus brevis Enterococus faecium Lactococcus lacs Lactobacillus buchneri Uncultured bacterium Uncultured bacterium Lactobacillus paracasei Lactococus buchneri Uncultured bacterium Uncultured bacterium Wickerhamomyces anomalus Kluyveromyces delphensis Kluyveromyces delphensis Candida glabrata Saccharum hydrid culvar Pichia kudriavzevii Saccharum hydrid culvar 1
2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 13
8
Figure 1 Bacterial and fungal communities of direct-cut and wilted elephant grass silages prepared with and without homo-fermentative and hetero-fermentative lactic acid bacteria inoculants and stored for 4 months in Hue, Vietnam. LP+LC, a mixture ofLactobacillus paraca- seiandLactococcus lactis;LB,Lactobacillus buchneri.
fermentative LAB inoculants, elimination ofK. delphensis may be necessary to obtain good aerobic stability. The antifungal acetic acid content of control silage may be insufficient to inhibit yeast activity during ensiling and after exposure to air; hence, LB inoculant can be consid- ered as a measure to ensure aerobic spoilage.
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