Selection of lactic acid bacteria from spontaneously fermented corn silage for the formulation of an inoculant

BORGO, M.F.; VINDEROLA, G.; BINETTI, A.; REINHEIMER, J.; BURNS, P.

Simposio; Simposio Internacional de Bacterias Lácticas; 2013

Silaging is the main method for preserving forages. A rapid drop in pH, caused by fermentation during ensiling, inhibits the growth of microorganism that causes silage spoilage. Lactic fermentation can occur spontaneously by the microflora naturally present in crops (with the risk of parallel butyric and acetic fermentation) or may be directed towards lactic acid fermentation by the use of inoculants, composed mainly of lactic acid bacteria (LAB) and enzymes. The use of inoculants standardizes the final product and produces sweet silage which is very palatable, resulting in higher intakes by the cattle and higher milk production. In our country, the use of inoculants for corn or alfalfa silage is widely spread; however, the vast majority of the commercial products available are not produced here. The aim of this study was to isolate and characterize lactic acid bacteria from spontaneous corn silage fermentation in order to formulate silage inoculants. Two samples of corn silage (where no inoculant was used) were obtained from farms located in Recreo and Esperanza (Santa Fe, Argentina) at days 0, 7, 14 and 21 of silage making. Serial dilutions of samples were made in peptone water and plated on MRS, M17, MRS-bile and MRS-LP agar (34°C, 48 h, anaerobiosis). Presumptive LAB isolates were frozen at -20°C and -70°C in MRS + glycerol (20%) and identified by 16S rRNA sequencing. Growth kinetics in MRS broth (30, 34, 37 and 43ºC), heat resistance to 55ºC for 5 and 15 (for selection of candidates for spray-drying) and the capacity to grow in corn silage extract (corn silage:water, 1:9, homogenized, filtered and autoclaved) (CSE), were determined. A total of 11 strains were isolated and identified as Lactobacillus plantarum Ls71, L. buchneri Ls141, L. amylovorus Hv142, L. amylovorus Hv212, L. amylovorus Hv214, L. fermentum Hv76, L. plantarum Hv75, L. panis Hv71, L. panis Hv73, L. panis Hv77 and Pediococcus acidilactici Ls72. Out of the temperatures assessed, best growth was observed at 34ºC and 37ºC. L. plantarum strains showed the fastest growth kinetics in MRS broth. Both the capacity to survive at 55ºC and the capacity to growth in CSE were strain dependent. P. acidilactici Ls72 showed the highest resistance to 55°C among tested strains (cell death was 0.2 log orders after 5 min) while L. buchneri Ls141, L. plantarum Hv75 and L. amylovorus Hv212 showed a cell death of 3.8, 2.7 and 2.2 log orders, respectively. When grown in CSE (34ºC, 24 h, initial inoculum 1% v/v), L. buchneri Ls141 showed a cell growth of 2.1 log orders whereas L. amylovorus Hv212, L. plantarum Hv75 and P. acidilactici Ls72 presented a cell growth of 1.2, 0.8 and 0.07 log orders, respectively. According to growth kinetics in MRS, growth in CSE and heat resistance, L. plantarum Hv75, L. buchneri Ls141 and P. acidilactici Ls72 were selected for further future studies of resistance to freeze and spray-drying for the formulation of an inoculant for silage fermentation.