Effect of Lactobacillus plantarum

Transcription

1 Effect of Lactobacillus plantarum Starter Culture on the Microbial Succession, Chemical Composition, Aerobic Stability and Acceptability by Ruminant of Fermented Panicum maximum Grass Ayodele Timi Adesoji *, Adeniyi Adewale Ogunjobi **, Obasola Ezekiel Fagade *** and Olaniyi Jacob Babayemi 1 Department of Botany and Microbiology, University of Ibadan, Ibadan, Nigeria <*timmyayus2002@yahoo.com; **aaogunjobi@hotmail.com; ***sekiteri2002@yahoo.com> Abstract In this study, starter culture of Lactic acid Bacteria (LAB) was developed by screening 20 different isolates of LAB to select the best that was used to inoculate P. maximum grass for silage production. The grass was inoculated with 1x10 6 cfu/ml of L. plantarum that grow over a wide range of ph and produce minimal concentration of lactic acid in de Man, Rogosa and Sharpe (MRS) broth. The fermented grass was sampled after 15, 30, 45 and 60 days of ensiling to determine microbial succession and chemical properties. Aerobic stability test of the silage was determined at 45 and 60 days of ensiling. The acceptability of the silage was also determined feeding of the silage to West African dwarf sheep. The LAB in the silage was observed to increase from 9.30 log cfu/g at day 15 to log cfu/g at day 45. This resulted in a rapid drop in the ph of the inoculated silage (decreasing from 4.50 at day 15 to 3.83 at day 30) compared to the uninoculated silage (that changed from 4.60 at day 15 to 4.73 at day 30). Mould and yeast was observed in the silage to be inhibited with mould count decreasing from 3.97 at day 15 to 2.46 at day 45 in the inoculated silage. During aerobic stability test, the increase in the count of spoilage microorganism was observed to be more drastic in the inoculated silage with yeast count increasing from 3.16 log cfu/g at day 1 of exposure to 3.56 log cfu/g at the second day at day 45 of ensiling. The ruminant fed the silage accepted the control silage more at days 45 and 60 with COP of 1.08 and 1.33, respectively. The CP of the inoculated silage increased from 7.20 g/100 DM at day 15 of ensiling to 9.84 g/100 DM at the end of day 60 of ensiling. The ADF and NDF were also reduced in the inoculated silage with g/100 DM of NDF for the control silage at day 30 and g/100 DM at the same day for the inoculated silage. It is, therefore, concluded that inoculation of silage with homolactic LAB increases fermentation of silage but not the aerobic stability of the silage. Keywords: Silage, sheep, Lactobacillus plantarum, fermentation, inoculum. Introduction Ensiling is an ancient method used to preserve the nutritive value of forages by packing and storing forage in airtight conditions. Silage fermentation occurs naturally under anaerobic conditions (the absence of oxygen). 1 Department of Animal science, University of Ibadan, Ibadan, Nigeria. <oj.babayemi@mail.ui.edu.ng> Plants contain native lactic acid bacteria (LAB), and when silage is placed under anaerobic conditions, these LAB produce lactic acid, reducing the ph to a level in which other bacteria cannot survive. However, LAB are not the only microorganisms on plants. Bacteria like Clostridia and Enterobacteria, as well as yeast and molds, also are present on plants and compete with LAB for sugars (Pahlow et al. 2003). Silage exposed to air eventually deteriorates as a result of aerobic microbial activity (Jonsson 1989). The factors that Regular Paper 11

2 influence deterioration include oxygen (exposure time), composition of the microbial population, substrate type, and temperature (Jonsson 1989). Lactate-assimilating yeast (Saccharomyces, Candida, Cryptococcus, and Pichia spp.) are usually the initial cause of aerobic deterioration (Ranjit and Kung 2000). Some strains of lactate-assimilating yeast grow well in an environment in which the ph level is between 3 and 8 and the temperature is under 40 C (Jonsson 1989). Some aerobic microorganisms can be harmful to livestock. Therefore, the prevention of aerobic deterioration of silage is important (Cai et al. 1999). As lactic acid and other residual sugars are assimilated by yeast, the temperature starts to rise (Jonsson 1989). Once the temperature is above 45 C, the amount of yeast present declines and other microbial organisms (bacilli) begin to accumulate. As aerobic deterioration takes place, there are changes in the chemical parameters of silage (Lindgren et al. 1985). The ph level tends to increase, ammonia and amines accumulate, and the levels of organic acids (lactic and acetic acid) tend to decline (Jonsson 1989). It is difficult to ensure good quality silage using tropical pasture crops because they usually have low water soluble carbohydrate (WSC) content, high buffering capacity, and low lactic acid bacteria (LAB) numbers (Catchpoole and Henzell 1971). These conditions result in low lactic acid production; in fact many researchers have found that silage made from tropical crops generally have high ph value and acetic acid content (Panditharatne et al. 1986; Imura et al. 2001). It is possible to apply bacterial inoculants at ensiling in order to promote adequate fermentation patterns. Inoculants, comprising homofermentative LAB such as Lactobacillus plantarum, Enterococcus faecium, and Pediococcus species, are often used to control the ensiling fermentation by rapid production of lactic acid and the consequent decrease in ph (Weinberg et al. 1993; Filya et al. 2000, Filya 2002a,b). Therefore, the objective of this experiment is to screen different LAB isolates by carrying out different physiochemical tests on each of the isolates and selecting one out of the isolates to inoculate P. maximum silage and determine its effects on the physical, chemical, microbial, aerobic stability of silage and acceptability by ruminant animals. Culture Medium Materials and Methods In this study, de Man, Rogosa and Sharpe (MRS) medium (de Man et al. 1960) was used for the physiochemical screening. Growth at Different Temperatures A concentration of 2.8 x 10 8 cfu in 0.1ml suspension of LAB species was inoculated into sterilized MRS broth. Incubation was then carried out at 25 C, 35 C, 37 C, and 45 C for 48 hours. The cell growth was monitored by measuring the absorbance at 570 nm using a spectrophotometer (Spectrumlab 752S). Growth at Different ph A concentration of 2.8 x 10 8 cfu in 0.1 ml suspension of LAB species was inoculated into sterilized MRS broth whose ph has been adjusted to 2, 4, 6 and 8 with phosphate buffer solution and incubated for 48 hours. Growth was monitored by measuring the absorbance at 570 nm with a spectrophotometer (Spectrumlab 752S). Growth at Aerobic and Aanaerobic Environment LAB suspension containing 2.8 x 10 8 cfu in 0.1 ml was inoculated into sterilized MRS broth. Incubation was carried out in a microaerophilic environment at 37 C for anaerobic environment and incubation at 37 C in an incubator was carried out for the aerobic incubation. Growth was monitored with a spectrophotometer (Spectrumlab 752S) after 48-hour incubation. Test for Lactic Acid (g/l) Production in MRS Broth by Tested Isolates The organisms were grown in sterilized MRS broth incubated at 37 C in a micro- Regular Paper 12

3 aerophilic environment at 37 C for 48 hours. It was then centrifuged at 3,000 rpm for 15 min. The supernatant was then titrated with 0.25 moll -1 NaOH and 1 ml of phenolphthalein indicator (0.5% in 50% alcohol). The titratable acidity was calculated as lactic acid (%). Each milliliter of 1N NaOH is equivalent to mg of lactic acid. The titratable acid was then calculated according to AOAC method (1990): Titratable acidity = (Ml NaOH NaOH M.E. 100) / Volume of sample used, where: Ml NaOH = Volume of NaOH used; N NaOH = Normality of NaOH solution; M.E. = Equivalent factor. Preparation of Silage Inoculants Identified and characterized Lactobacillus plantarum from Panicum maximum silage from Environmental and Biotechnology Laboratory, Department of Botany and Microbiology, University of Ibadan, Ibadan, Oyo State, Nigeria, was inoculated into 500 ml of prepared MRS broth in Erlenmeyer s flask. It was then incubated on a shaker incubator for 2-3 days at 250 rpm and diluted with 2,500 ml of sterile distilled water. One milliliter was pipette pulled out to determine the acquired bacterial load by plate count on MRS agar using pour plate technique. Schedule of Ensiling Experiment Panicum maximum grass that was established at the teaching and research farm of the University of Ibadan, Ibadan, Oyo State, Nigeria, was harvested in the month of November 2008 after six weeks of re-growth. The grass was chopped to 2-3 cm size and wilted for 24 hours. The wilted grass was taken for chemical and microbiological analysis. The chopped grass was then divided into equal portions of 1 kg and packed into polyethylene bags and properly sealed to exclude air for anaerobic condition after inoculation with 10 ml of approximately 10 6 cfu/ml of the L. plantarum. The silage was then opened after 15, 30, 45 and 60 days for microbiological, chemical, aerobic stability and acceptability by ruminant. The control was an un-inoculated grass at each one of the days in which the silage was opened. The experiment was replicated 3 times to reduce errors. Microbial, Physical and Chemical Analysis of the Silage Exactly 1 g of the silage was mixed with 9 ml of sterile distilled water at each day of opening the silage. Serial dilution was carried out within 10-6 to The dilutions were then plated on the following media: de-man- Rogosa-Sharpe agar (MRS) to detect LAB, Nutrient Agar (NA) to detect aerobic bacteria, Yeast extracts Agar (YEA) for yeast, Potato Dextrose Agar (PDA) to detect Mould and Eosin Methylene Blue (EMB) for coliforms. Coliform was incubated at 37 C for 1-2 days, as well as a MRS plate at 37 C in a microaerophilic environment for 2 days. Aerobic bacteria plate was incubated at 37 C for 1-2 days, Mould and Yeast plates were cultured at 25 C for 3 days. Numbers of colony forming (cfu) were expressed in log cfu/g. The temperature of the silage was measured with a thermometer. Then 25 g of the silage was taken from the silage at each of the days of opening it, mixed with 100 ml of distilled water and shaken on a shaker at 250 rpm for 15 min. It was then filtered with a filter paper and the filtrate was used for the determination of the ph with a ph meter. Dry matter ( content of the silage was determined by oven drying at 80 C for 48 hours. Ash, crude fibre, and ether extract content of the silage were determined according to AOAC (1990). Neutral detergent fibre (NDF), acid detergent fibre (ADF) and acid detergent lignin (ADL) were determined according to Goering and van Soest (1970), crude protein (CP) content of the silage was also determined by the Kjeldahl method (AOAC 1990). Aerobic Stability Test At the end of days 45 and 60 of ensiling, the silage was subjected to aerobic stability test that lasted for 7 days. Samples of the silage were placed in a petri dish and placed in dessicators. Samples were taken at days 1, 2, 5, and 7 for the microbiological analysis of the Regular Paper 13

4 silage. The count of the LAB, yeast, mould, aerobic bacteria and coliforms were determined as mentioned above. Acceptability by Ruminant Eight West African dwarf sheep weighing kg and about two years old were used to evaluate the free choice intake of the silage (control and LAB treated) at each of the days of uncovering the silage, i.e., days 15, 30, 45 and 60, respectively. Exactly 2.5 kg of each silage, both the LAB treated silage and the control, was placed in strategic feeding troughs measuring 1 m x 2 m. The sheep were allowed to feed for 6 hours per day on each day of the feed out. Consumption was measured by deduction of remnants from the amount of silage served after 4 hours. The silage preferred was assessed from the coefficient of preference (COP) value, calculated from the ratio between the intake for the individual silage treated with LAB or control divided by the average intake of the silage treated with LAB or control (Babayemi et al. 2006). On this basis, a silage treatment was inferred to be relatively acceptable if COP was greater than unity. Statistical Analysis of Silage The data were analyzed by analysis of variance and means were separated by Duncan s multiple range (Duncan 1955). Results Physiochemical Screening of LAB Isolates Growth of eighteen different isolates of Lactobacillus plantarum and two isolates of Lactobacillus brevis at different temperature, ph, salt, and concentration was carried out on MRS broth. Production of lactic acid in MRS broth and growth in anaerobic and aerobic condition for each of the isolates was also carried out. The results of these tests are shown in Tables 1, 2, 3 and 4, respectively. Organism 60, which was found to grow over a wide range of ph by having the highest optical density of at ph 8 out of all the isolates tested, was used for the Panicum maximum fermentation. Table 1. Growth of LAB in MRS broth at different temperatures. Organisms 25 o C 35 o C 37 o C 40 o C 45 o C Note: All the numbers on the organisms column represents L. plantarum except numbers 33 and 45 which represent L. brevis. Table 2. Growth of LAB isolate in MRS broth at different ph. Organisms ph 2 ph 4 ph 6 ph Microbial Succession in Silage at Different Days of Ensiling The results of microbial succession in silage at different days are shown in Table 5. Regular Paper 14

5 Table 3. Growth of LAB isolates in MRS broth at different NaCl concentration. Organisms 3% 5% 7% 9% Note: All the numbers on the organisms column represents L. plantarum except numbers 33 and 45 which represent L. brevis. Table 4. Quantity of lactic acid produce and growth of LAB isolates in different O 2 regimes. Organisms Titrable lactic acid Anaerobic Aerobic Note: All the numbers on the organisms column represents L. plantarum except numbers 33 and 45 which represent L. brevis. Table 5. Microbial succession in silage at different days of ensiling and microbial count on fresh unensiled wilted P. maximum (log cfu/g). Total aerobic bacteria Days of ensiling Control 7.95 a 7.94 a 4.94 b 4.95 a Org d 3.94 c 2.94 c 2.98 b Total aerobic bacteria count on fresh grass 3.6 Control 4.30 b 4.30 a 3.65 c 3.84 d Coliform Org b 4.24 a 4.34 b 4.38 b Coliform count on fresh wilted P. maximum 5.0 Control 8.20 a 9.84 a 8.46 c 7.48 d LAB Org a 9.48 b a b LAB count on fresh wilted P. maximum 5.0 Control 4.00 c 4.00 b 4.16 b 4.27 a Yeast Org c 3.94 b 3.16 c 3.28 b Yeast count on fresh wilted P. maximum 5.3 Control 3.46 d 4.64 a 4.27 a 4.00 a Mould Org b 2.96 c 2.46 c 2.56 b Mould count on fresh wilted P. maximum 5.0 Note: Lactic acid bacteria (LAB), Org. 60 represent silage treated with L. plantarum. Values carrying different letters were significantly different at P < Significant difference (P < 0.05) occurs between the control and the inoculated silage in the total aerobic bacteria count in all the days of ensiling. At day 15, the count on the inoculated silage was 4.19 log cfu/g which was lower than that observed in the control where 7.95 log cfu/g was observed. Lower count was also observed in the silage as the days of ensiling increased in which 4.95 log cfu/g was observed in the control at day 60 while lower count of 2.98 log cfu/g was observed in the inoculated silage at the same day. Coliform count between the control and the inoculated silage did not show significant difference (P > 0.05) at days 15 and 30 of ensiling. The coliform count on the control silage was 4.30 log cfu/g at days 15 and 30 while that of the inoculated silage was 4.30 log cfu/g and 4.24 log cfu/g at days 15 and 30 of ensiling, respectively. LAB counts on the control and LAB treated silage are 8.20 log cfu/g and 9.30 log cfu/g, respectively, at day 15 of ensiling showing no significant difference (P < 0.05). The LAB count on the inoculated silage increased to log cfu/g and log cfu/g at days 45 and 60, respectively, while the Regular Paper 15

6 LAB count on the control silage decreased to 7.48 log cfu/g at the last day 60. The yeast count did not show significant difference (P < 0.05) at days 15 and 30. The yeast count on the control silage was 4.00 log cfu/g at days 15 and 30 while the yeast count on the inoculated silage are 4.00 log cfu/g and 3.94 log cfu/g at days 15 and 30 of ensiling, respectively. The yeast count decreased to 3.16 log cfu/g and 3.28 log cfu/g at days 45 and 60, respectively, in the inoculated silage while increase to 4.16 log cfu/g and 4.27 log cfu/g at days 45 and 60, respectively, was observed in the control silage. Significant difference was observed in the mould count between the control and inoculated silage in all of the ensiled days. The mould count decreased from 5.0 log cfu/g on the fresh wilted grass to 3.46 log cfu/g on the control silage and 3.97 log cfu/g on the LAB treated silage at day 15. Decrease to 2.56 log cfu/g was observed at day 60 in the inoculated silage while increase to 4.00 log cfu/g at day 60 was observed in the control silage. Physical Assessment and Chemical Composition of the Silage at Different Days of Ensiling These results of the physical and chemical composition of the silage at different days of ensiling are shown in Tables 6 and 7. The colour of guinea grass (silage) in all the LAB treated and untreated, i.e., the control silage, varies from olive-green to greenish yellow at 15, 30, 45 and 60 days of ensiling, respectively. The smell/tastes of 15 days silage were mild fruity smell to sharp fruity smell for all organism inoculated silages. Mild vinegar smell to sharp and very sharp vinegar odor/tastes were perceived for the guinea grass (control silage) and all the inoculated silage from days 30 to 60, respectively. The structures of guinea grass silage for all the silage were firm, visible and separable in the overall silage throughout the entire period of ensiling. Significant difference (P < 0.05) was observed in the ph of the silage at days 15 and 30 between the LAB treated and the control silage. ph of 4.60 and 4.73 at days 15 and 30, respectively, was observed in the control silage while decrease from 4.50 at day 15 to 3.83 at day 30 was observed in the inoculated silage. Significant difference (P < 0.05) was only observed at day 30 between the control and LAB treated silage in the temperature of the silage. The control at this day had temperature of 28 C while the LAB treated silage had a temperature of 25 C. Decrease from 29.6 C at day 15 to 25.3 C at day 60 in the temperature was observed in the inoculated silage. Significant difference (P < 0.05) was observed in the DM values in all the days of ensiling. The values of DM in the control silage range from between g/100 DM to g/100 DM while that of the inoculated silage range between g/100 DM to g/100g DM. The DM of the fresh wilted grass was 30.43g/100 DM. Crude protein (CP) of the control silage range from between 6.14 g/100g DM to 7.86 g/100g DM while CP of the inoculated silage was higher, increasing from 7.20 g/100g DM at day 15 to 9.84 g/100g DM at day 60. Table 6. Physical quality of silage by colour, structure and taste of silage. Days ensiled Treatments Parameters Control Org. 60 Colour Olive green Olive green Smell/ Mild fruity Very sharp tastes smell fruity smell Structure Firm and Firm and separable separable Colour Olive-green Greenish yellow Smell/ Sharp Vinegar tastes vinegar Structure Firm and Firm and separable visible Colour Olive green Olive green Smell/ Very sharp Sharp tastes vinegar vinegar Visible, firm Visible, firm Structure and and separable separable Colour Olive green Olive green Smell/ Mild Sharp tastes vinegar vinegar Structure Visible and Visible and separable separable Note: Org. 60 is the silage treated with the two L. plantarum. Regular Paper 16

7 Table 7. Physical and chemical composition of silage at different days of ensiling. ph Days of ensiling Treatments Control 4.60 a 4.73 a 3.90 a 2.90 a Org ab 3.83 b 3.90 a 3.03 a ph of fresh wilted grass 6 Control a a a b Temperature ( 0 C) DM CP EE Ash ADF NDF ADL CF Org a b a b Control a a a a Org b b b b DM of fresh wilted grass Control 6.13 c 7.44 c 7.86 d 6.78 c Org b 9.40 a 9.40 a 9.84 a CP of fresh wilted grass 6.56 Control a c c c Org b b a b EE of fresh wilted grass Control b c c 8.00 c Org b b a a Ash of fresh wilted grass 8.00 Control a a a a Org b b b b ADF of fresh wilted grass Control a a c c Org b bc a a NDF of fresh wilted grass Control 7.89 c 8.11 c 8.57 a 8.60 c Org b 8.33 b 8.33 b 8.82 b ADL of fresh wilted grass Control a a b b Org a a b a Note: Dry matter (, Crude protein (CP), Ether extract (EE), Acid detergent fibre (ADF), Neutral detergent fibre (NDF), Acid detergent Lignin (ADL), Crude fibre (CF), Org. 60 is silage inoculated with L. plantarum. Values carry different letters were significantly different at P < The Ether extract (EE) of the control silage range from between 11 g/100g DM to g/100g DM while that of the inoculated silage increased from g/100g DM at day 15 to g/100g DM at day 30 and decreased to g/100g DM at day 45. Significant difference (P < 0.05) was observed between the control and the inoculated silage in all the days of ensiling. The ash content of the silage showed no significant difference at day 15 between the control and the inoculated silage. Both silages at this day had g/100g DM ash content. Increase to g/100g DM was observed in the control, while increase to g/100g DM was observed in the inoculated silage at day 30. Decrease to 8.00 g/100g DM was observed in the control, while decrease to g/100g DM was observed in the inoculated silage at day 60 of ensiling. The ADF content of the silage was significantly different (P < 0.05) in all the days of ensiling. That of the inoculated silage was observed to be lower in all the days of ensiling having values of g/100g DM, g/100g DM, g/100g DM and g/100g DM at days 15, 30, 45 and 60, respectively, while that of the control had values of g/100 DM, g/100 DM, g/100 DM and g/100 DM at days 15, 30, 45 and 60, respectively. The NDF content was also observed to be significantly different (P < 0.05) between the control and the inoculated silage in all the days of ensiling. At days 15 and 30 the NDF content of the control silages was g/100 DM and g/100 DM, while that of the LAB treated silage had values of g/100 DM and g/100 DM at days 15 and 30, respectively. The ADL values of the control range from between 7.89 g/100 and 8.60 g/100 in all the days of ensiling while that of the inoculated silage range between 8.33 g/100 DM and 8.82 g/100 DM in all the days of ensiling. Significant difference was observed only at day 60 of ensiling in the crude fibre (CF) content of the silage between the control and the inoculated silage in which the control had values of g/100 DM and the inoculated silage had values of g/100 DM at this day. Microbial Succession in Silage after Exposure to Air at 45 Days of Ensiling The results of the microbial succession after exposure of the silages to air at 45 days of ensiling are shown in Table 8. The count of the Regular Paper 17

8 aerobic bacteria in both the control silage and the inoculated silage increased as the days of exposure to air increased. The highest increase was recorded at day 5 of exposure in which the control silage had values of 9.84 log cfu/g, while the inoculated silage had values of 8.74 log cfu/g. The LAB count decreased from log cfu/g at day 1 of exposure to 3.81 log cfu/g at the end of the seventh day of exposure. In the control, silage decreased from 8.46 log cfu/g at day 1 of exposure to 4.87 log cfu/g at day 5 of exposure. The yeast count in the control silage decreased to 2.17 log cfu/g at day 2 from 4.16 log cfu/g at day 1 and increase to 5.64 log cfu/g was recorded at the fifth day of exposure. The yeast count in the inoculated silage increased drastically, as the days of exposure to air increased, from 3.16 log cfu/g at day 1 to 5.46 log cfu/g at the 5 th day of exposure. The mould count, which is also an indication of spoilage, also increased drastically in the inoculated silage, as the days of exposure increased, from 2.46 log cfu/g at day 1 to 6.57 log cfu/g at the end of the 7 th day of exposure. Microbial Succession in Silage after Exposure to Air at 60 Days of Ensiling The result of the microbial succession at 60 days of ensiling is shown in Table 9. A similar succession was also observed here. The count of aerobic bacteria increased from 4.95 log cfu/g at day 1 to 9.24 log cfu/g at the end of day 7 of exposure in the control silage. In the inoculated silage, the count increased from 2.98 log cfu/g at day 1 to 4.96 log cfu/g at the end of the 5 th day of exposure. In the control silage, the LAB count decreased from 7.48 log cfu/g at day 1 of exposure to 3.98 log cfu/g at day 7 of exposure. In the inoculated silage, the count decreased from log cfu/g at day 1 to 5.94 log cfu/g at the end of the 7 th day of exposure. The yeast count in the control silage decreased from 4.27 log cfu/g at day 1 of exposure to 2.64 log cfu/g at day 2 of exposure and increased to 6.98 log cfu/g at the end of the 7 th day. In the inoculated silage, increase from 3.28 log cfu/g at day 1 to 7.90 log cfu/g at the end of the 7 th day was observed. The mould count also decreased in the control silage to 2.54 log cfu/g at day 2 from 4.00 log cfu/g at day 1, and then increased to 6.46 log cfu/g at the end of day 7 of exposure. In the inoculated silage, increase from 2.54 log cfu/g at day 1 to 7.89 log cfu/g at the end of day 7 was observed. Table 8. Microbial succession in silage after exposure to air at 45 days of ensiling (log cfu/g). LAB Yeast Mould Control 8.46 c 7.84 c 4.87 c 3.64 c Org a 8.75 a 5.28 a 3.81 b Control 4.16 b 2.17 d 5.64 b 4.74 c Org c 3.56 b 5.46 c 4.68 c Control 4.27 a 2.01 b 5.90 b 6.89 a Org c 3.89 a 5.95 ab 6.57 b Note: Lactic acid bacteria (LAB), Org. 60 is silage inoculated with L. plantarum. Values carrying different letters were significantly different at P < Table 9. Microbial succession in silage after exposure to air at 60 days of ensiling. Days of exposure to air Treatments Aerobic Control 4.94 b 4.25 d 9.84 a 6.59 a bacteria Org c 4.74 c 8.74 c 3.94 ab Days of exposure to air Treatments Aerobic Control 4.95 a 4.98 b 8.74 a 9.24 a bacteria Org b 4.89 b 4.96 b 4.57 d LAB Yeast Mould Control 7.48 d 6.94 d 4.84 b 3.94 d Org b 9.56 b 6.58 a 5.94 a Control 4.27 a 2.64 d 6.84 a 6.98 b Org b 3.48 c 4.96 c 7.90 a Control 4.00 a 2.54 d 5.74 a 6.46 d Org b 2.84 b 4.56 b 7.89 a Note: Lactic acid bacteria (LAB), Org. 60 is silage inoculated with L. plantarum. Values carrying different letters were significantly different at P < 0.05 sil. Regular Paper 18

9 Acceptability of Inoculated and Uninoculated P. maximum Silage by West African Dwarf Sheep The acceptability results are shown in Table 10. The coefficient of preference (COP), which is used to determine the acceptability of the silage by the ruminant, had the same value of 0.99 at days 15 and 30 for the inoculated silage, which decreased to 0.92 at day 45 and 0.67 at day 60 of ensiling. The COP for the control was higher, having values of 1.01, 1.00, 1.08 and 1.33 at days 15, 30, 45 and 60, respectively. The MDI for the control silage range between 2.02 and 2.20, while that of the inoculated silage range between 1.04 and Table 10. Acceptability of organism treated and untreated P. maximum silage by West African dwarf sheep. Days of ensiling and acceptability Treatment Control Org MDI COP MDI COP MDI 45 COP MDI COP Note: (MDI)(kg mean daily matter intake and (COP) coefficient of preference. Org. 60 is the silage inoculated with the L. plantarum. Discussion The selection of the best strains is essential and should be based on their safety and their ability to grow over a wide range of dry matter contents, temperature and ph values, to utilize the sugar found in forages, and their ability to rapidly reduce the number of contaminating bacteria and fungi (Seale 2006). This is why the isolates of L. plantarum used for the silage inoculation were first subjected to the physiochemical test in order to get the best out of the isolates. In all the silage, the greenish colour of the silage is retained and the odour and texture of the silage is maintained by that adopted by Breirem and Ulvesli (1960). All the silage formed in this experiment was a good quality silage from appearance and odour. Homolactic Regular Paper 19 bacteria have been used to inoculate silages and improve their fermentation (Kung et al. 1984; Stokes 1992). Inhibition of bacteria (via low ph) and fungi (via the elimination of oxygen) in the process of silage fermentation are the key factors that are responsible for making high-quality silage that is stable when exposed to air. Addition of homofermentative LAB to forage at ensiling can enhance the fermentation process by producing high concentrations of lactic acid and rapidly reducing the ph of silage. Theoretical homolactic fermentation of 1 mole of hexose yields 2 moles of lactate with a DM recovery of 100% and an energy recovery of 99% (McDonald et al. 1991). Heterolactic fermentation results in energy losses of 1 to 2%, but the DM losses vary depending on the substrate and pathway used. Specifically, fermentation of fructose results in a 5% loss of DM, but fermentation of glucose results in a 24% loss of DM. In recent years, treating silages with L. plantarum strain MTD1 has improved DM intake and milk yield in a number of studies (Kung and Muck 1997). Therefore, the homolactic L. plantarum carefully selected for this study was able to inhibit the growth of spoilage mould, yeast and aerobic bacteria in the inoculated silage. The inhibition was observed at day 30 of ensiling to be more than in the uninoculated silage. At this day, the count of the aerobic bacteria, yeast, and mould were 3.94 log cfu/g, 3.94 log cfu/g and 2.96 log cfu/g, respectively. These counts were observed to decrease more in the inoculated than in the uninoculated silage as the days of ensiling increase. Many studies (Cai and Kumai 1994; Cai et al. 1997) have reported that lactobacilli are the dominant microbial population on forage crops and contribute to silage fermentation. The lactobacilli play a more important role in fermentation processes and effectively promote lactic acid fermentation for a longer time than do lactic acid-producing cocci (e.g., enterococci, streptococci, leuconostocs, weissella, and pediococci). Generally, when the lactobacilli reach at least 10 5 cfu/g of FM, silage can be well preserved (Hellings et al. 1985). The addition of LAB inoculants at ensiling is intended to ensure rapid and vigorous fermentation that results in faster

10 accumulation of lactic acid, lower ph values at earlier stages of ensiling, and improved forage conservation. Many studies (Cai and Kumai 1994; Cai et al. 1997; Hellings et al. 1985; McDonald et al. 1991; Sanderson 1993; Sebastian et al. 1996; Sharp et al. 1994; Weinberg et al. 1993) have shown the advantage of such inoculants. Therefore, the L. plantarum used in this study was able to reduce the ph of the silage rapidly by bringing the ph down to as low as 3.83 within the first 30 days of ensiling compared to the control that had ph of 4.73 at the same day. This indicates a rapid and vigorous fermentation in the inoculated silage compared to the uninoculated control. The increase in the count of the LAB in the inoculated silage as the days of ensiling increase is also another indication of a good fermentation in the inoculated silage compared to the uninoculated silage. Although the lactic acid of the silage is not determined, it could have been higher at the beginning of the ensiling in the inoculated silage than the uninoculated control silage. Generally, alfalfa has relatively high lactic acid buffering capacity and protein content, but low WSC content, and numbers of lactobacilli (Cai et al. 1994; and McDonald et al. 1991). During silage fermentation, the lactobacilli could not produce sufficient lactic acid to inhibit the growth of clostridia. As a result, the ph value of alfalfa silage did not decline to < 4.2, allowing butyric fermentation by clostridia to occur. However, Cai et al. (1999) in their experiment with Alfafa, Italian ryegrass and Sorghum reported ph of 5.6 for uninoculated Alfafa silage and 5.0 with Alfafa silage inoculated with L. casei and L. plantarum. They also reported ph of 5.0 for uninoculated Italian ryegrass and 4.2 and 4.5 for Italian ryegrass inoculated with L. casei and L. plantarum, respectively. In their study with Sorghum, silage ph of 3.8 and 3.7 was reported for silage inoculated with L. casei and L. plantarum, respectively. All these ph were reported after 40 days of ensiling. This lower ph of Sorghum and higher in Alfafa and Italian ryegrass silage could be as a result of higher lactic acid buffering capacity and protein content and lower WSC content of the silage compared to that of the Sorghum silage. This could also be responsible for the lower ph of 3.83 observed in this work with the inoculated silage at day 30, which went to as low as 3.03 at the end of day 60 of ensiling. When the silo is opened, aerobic conditions prevail at feeding time, and the silage is subject to aerobic microbial growth and is potentially unstable (Ohyama and Hara 1975; Ohyama et al. 1975; and Weinberg et al. 1993). Some aerobic microorganisms can be harmful to livestock. Therefore, the prevention of aerobic deterioration of silage is important (Cai et al. 1999). However, the inoculation of silages with homofermentative LAB, such as L. plantarum used in this study, has not always resulted in silage with good aerobic stability (Kung et al. 1991; Sanderson 1993). In a review of the literature, (Muck and Kung 1997) reported that inoculation improved aerobic stability in less than 30% of the studies reported between 1990 and Silages treated with microbial inoculants had no effect on the aerobic stability or made it worse in the remaining studies. The two main reasons for these findings include the fact that relatively acid-tolerant yeasts that assimilate lactate (and usually not bacteria) are primarily responsible for spoilage of silages when exposed to air. Secondly, lactic acid by itself is not an effective antimycotic agent (Moon 1983; and Woolford 1975). This probably may be the reason why the count of opportunistic aerobic bacteria, spoilage yeast and mould in the experiment increased rapidly as the silage in this experiment was exposed to air. The more lactic acid in the inoculated silage probably could have also resulted in rapid increase in the number of mould and yeast in the inoculated silage compared to the uninoculated control. Cai et al. (1999) reported that most silage yeasts were able to grow at low ph conditions and assimilated lactic acid during the aerobic exposure. The authors then concluded that lactobacilli improve fermentation quality but did not inhibit the growth of yeast and might have increased aerobic deterioration of the silage. The L. plantarum used in this study was observed to increase the CP and reduce the NDF and ADF of the silage. This is in contrast to what was reported by Kung et al. (2007) Regular Paper 20

11 where no difference was detected among treatment means for the final concentration of CP, ADF, NDF or WSC, when high moisture corn was ensiled with L. buchneri 40788, a mixture of enzymes, L. buchneri and a mixture of enzymes, and a liquid mould inhibitor for 90 days. Similar result has also been reported by Kleinschmit and Kung (2006), that inoculation did not affect the concentrations of CP, NDF, or ADF of ensiled forage crops. However, they did report that inoculation resulted in a trend for lower concentrations of WSC. But the result of this experiment is similar to what was reported by Kung and Ranjit (2001), in which lower ADF and NDF concentrations were observed compared to untreated silage, when the authors treated Barley silage with various blends of homolactic acid bacteria, propionibacteria, and enzymes. Soderholm et al. (1988) reported that crude protein content of high moisture corn is relatively low and can be increased by addition of NPN at ensiling. Although the authors reported that some information (Britt and Huber 1976; Dutton and Otterby 1971; Mowat et al. 1981) is available on the addition of NPN to corn grain, it has not gained wide acceptance by livestock producers. One major reason the authors emphasis is that large amounts of free NH 3 have been detected in treated corn, which reduced acceptability to cattle (Mowat et al. 1981). Therefore, the LAB inoculants used in this study are quite important, because of their effects in increasing the crude protein of the silage as well as being a biological additive which is widely acceptable in the world as they are safe and easy to use, non-corrosive to machinery, they also do not pollute the environment and are regarded as natural products (Filya et al. 2000). Weinberg et al. (2001) and Rodríguez et al. (1998) demonstrated that higher temperatures promote microbial activities and negatively affect the fermentation process and aerobic stability. In this study, the decrease in the temperature of the silage as the days of ensiling increase could be an advantage for the fermentation process in the silage. Kim and Adesogan (2006) reported that high ensiling temperature and simulated rainfall had detrimental effects on the fermentation process and silage quality. The authors therefore recommended that corn silage producers in hot, humid regions need to adhere strictly to excellent silage-making practices to overcome the adverse effects of moisture and temperature on corn silage production. It has been shown that the infusion of lactic acid into the rumen (Dowden and Jacobson 1960; Montgomery et al. 1963), or supplementing the ration with lactic acid (Bentley et al. 1956; Ekern and Reid 1963; Emery et al. 1961; Johnson et al. 1962; Montgomery et al. 1963; Thomas et al. 1961), may cause a depression in voluntary intake of dry matter in animals fed hay (Bentley et al. 1956; Emery et al. 1961; Johnson et al. 1962; Montgomery et al. 1963; Thomas et al. 1961). The depression was usually less than that observed when silage was fed, although the amount of lactic acid added was similar to the quantities which would be ingested from feeding silage. This was similar to what was observed in this study with the ruminants, i.e., West African dwarf sheep used in this study for the acceptability studies. Almost the same COP at days 15 and 30 of ensiling was observed, but as the days of ensiling increased to 45 and 60, the COP of the inoculated silage decreased while that of the uninoculated silage increased. Therefore, it could be as a result of increase in the concentration of lactic acid in the inoculated silage compared to the uninoculated silage as the days of ensiling increased. Conclusion Inoculation of silage with homolactic LAB should be encouraged in that it was able to result in rapid decrease of ph of the silage, rapid increase in LAB and inhibition of spoilage microorganism. Hence, good fermentation of silage was ensured. The inoculation also results in decrease in the ADF and NDF of the silage and increase in the CP content of the silage. But if the producer is interested in aerobic stability of silage inoculation with homolactic LAB, like L. Plantarum used in this study, it should be discouraged as suggested by Contreras-Govea et al. (2009). Regular Paper 21

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