Agricultural and Food Science, Vol. 15 (2006): 185–199. 185 A G R I C U L T U R A L   A N D   F O O D   S C I E N C E Vol. 15 (2006): 185–199. © Agricultural and Food Science Manuscript received June 2006 Effect of lactic acid bacteria inoculants, formic acid,  potassium sorbate and sodium benzoate on  fermentation quality and aerobic stability of  wilted grass silage Eeva Saarisalo, Taina Jalava MTT Agrifood Research Finland, Animal Production Research, FI-31600 Jokioinen, Finland, e-mail: eeva.saarisalo@mtt.fi Eija Skyttä, Auli Haikara VTT Technical Research Centre of Finland, PO Box 1000, FI-02044 VTT, Finland Seija Jaakkola MTT Agrifood Research Finland, Animal Production Research, FI-31600 Jokioinen, Finland, present address: Department of Animal Science, PO Box 28, FI-00014 University of Helsinki, Finland The efficiency of a novel strain of lactic acid bacteria inoculant (Lactobacillus plantarum VTT E-78076, E76) on the fermentation quality of wilted silage was studied. Furthermore, the possibility to improve aero- bic stability of silages by combining an inoculant and chemical preservatives was investigated. Two ex- periments were conducted with wilted timothy-meadow fescue herbage (dry matter 429 and 344 g kg-1) using six treatments. In experiment I, E76 (106 cfu g-1 fresh matter (FM)) was applied alone and in combina- tion with sodium benzoate (0.3 g kg-1 grass FM) or low rate of formic acid (0.4 l t-1 FM). In experiment II, E76 and a commercial inoculant were applied alone and in combination with sodium benzoate. Untreated silage and formic acid (4 l t-1 FM) treated silage served as negative and positive controls in both experi- ments. The effect of sodium benzoate and potassium sorbate in experiment I, on aerobic stability was tested by treating silages prior to aerobic stability measurements. The novel lactic acid bacteria inoculant was equally effective in improving fermentation quality as the commercial inoculant. However, the aerobic stability of both inoculated silages was poorer than that of formic acid treated or the untreated one in one of the experiments. The results suggested that antimicrobial properties of E76 were not effective enough to improve aerobic instability. One option to overcome this problem is to use chemical additives in combina- tion with the inoculants. Key words: silage making, grass silage, wilting, additives, aerobic stability, inoculants, formic acid, fermen- tation, sodium benzoate, potassium sorbate 186 A G R I C U L T U R A L   A N D   F O O D   S C I E N C E Saarisalo, E. et al. Additives to improve aerobic stability of wilted silage Introduction Biological and chemical silage additives are used primarily to improve the fermentation quality and are therefore considered more useful in low dry matter (DM) silage. Direct acidification with an organic acid, for example with formic acid, re- stricts fermentation i.e increases residual water soluble carbohydrates (WSC) and decreases pro- duction of volatile fatty acids (VFA) and protein degradation (Chamberlain and Quig 1987, Jaak- kola et al. 1991, 2006). The improved fermenta- tion quality increases silage intake, microbial protein synthesis in the rumen and animal pro- duction (Huhtanen et al. 2002, 2003). Increasing the DM content of the herbage by wilting restricts the silage fermentation due to the lower water ac- tivity. Lactic acid bacteria (LAB) inoculants can be used to improve the silage fermentation quality in- stead of corrosive and hazardous acid based addi- tives. Lactobacillus plantarum (VTT E-78076, E76) is known for its broad-spectrum antimicro- bial activity both against gram positive and gram negative bacteria and, furthermore, against Fusar- ium moulds (Haikara et al. 1993, Haikara and Laitila 1995, Niku-Paavola et al. 1999, Laitila et al. 2002). Despite being originally isolated from beer, the L. plantarum E76 strain proved to be a potential grass silage inoculant in a screening pro- cedure (Skyttä et al. 2002a) and in a laboratory ensiling trial (Saarisalo et al. 2006). It was efficient in producing lactic acid, lowering pH rapidly and decreasing the ammonia-N production. Although efficient in improving silage fermentation, homo- fermentative LAB inoculants have sometimes im- paired the aerobic stability (Weinberg et al. 1993, Weinberg and Muck 1996). Increasingly herbage is wilted prior to ensiling in northern Europe mainly due to technological advantages. Dryer herbage decreases risk of poor fermentation quality estimated by the amount of VFA and proportion of ammonia-N of nitrogen (Jonsson et al. 1990, Yan et al. 1998, Field et al. 1999). However, with increasing DM, problems associated with aerobic stability tend to increase (Wyss 1999). Yeasts capable of utilising lactate as the source of energy start the aerobic deterioration (Woolford 1990, McDonald et al. 1991). The de- crease in lactate increases the silage pH which im- proves growth conditions for the microbes, deteri- orating the nutritional and hygienic quality of si- lage. The aerobic catabolism of nutrients generates heat, and therefore, an increase in silage tempera- ture is a good indicator for the growth of microbes and nutrient losses. Although the role of additives in controlling fermentation is less important in wilted material, there might be an additional ben- efit in using additives in high DM silages in order to improve aerobic stability. Hurdle technology could provide an alternative tool for controlling growth of yeasts in silage since their growth cannot be inhibited by any single en- vironmental parameter. Preservation by hurdle technology is based on simultaneous application of various antimicrobial factors affecting the target organisms by different mechanisms (Leistner and Gorris 1995). In ensiling, either a combination of synergistically working inoculants (Rauramaa et al. 1996, Driehuis et al. 1999, Weinberg et al. 1999), or a combination of an appropriate inocu- lant and chemical treatment (Beck 1989 ref. by Weissbach et al. 1991) could be considered. Skyttä et al. (2002b) showed that a combination of a se- lected lactic inoculant, potassium sorbate and so- dium benzoate inhibited in vitro the growth of four spoilage yeast strains isolated from grass silage [Pichia anomala (lactate+) VTT C-00352, Toru- laspora delbrueckii (lactate+) VTT C-00355, Rho- dotorula mucilaginosa (lactate-) VTT C-00353 and Pichia kluyveri var. kluyeveri (lactate-) VTT C-00354]. The first aim of the experiments was to study the effectiveness of the LAB inoculant E76, previ- ously selected (Skyttä et al. 2002a) and tested in the laboratory environment (Saarisalo et al. 2006), in less controlled i.e. more practical and demand- ing conditions, and to compare it with a commer- cial LAB inoculant. Another object was to study the possibilities to improve the aerobic stability of wilted silage by applying the hurdle technology. The preliminary results of experiment I have been presented by Saarisalo et al. (2001). 187 A G R I C U L T U R A L   A N D   F O O D   S C I E N C E Vol. 15 (2006): 185–199. Material and methods Material and treatments Two trials were carried out at MTT Agrifood Re- search Finland, Jokioinen, using pilot scale silos (1 m3) made of metallic cylinders and lined with plastic. Both experiments consisted of six treat- ments (Table 1). In experiment I, silages were pre- pared from second cut timothy (Phleum pratense) -meadow fescue (Festuca pratensis) grass, mown with a mower conditioner, wilted for 6 h and lifted with a precision-chop forage harvester. Treatments were: 1) Untreated (UT), 2) Formic acid 4 l t-1 FM, [5 l t-1 AIV2Plus (formic acid 760 g kg-1, ammo- nium formate 55 g kg-1, Kemira Oyj, Finland), FA4], 3) L. plantarum VTT E-78076 inoculant, freshly cultured, 1 × 106 cfu g-1 FM (E76), 4) E76 + a low rate of formic acid (AIV2Plus, 0.4 l t-1, FA0.4), (E76 + FA0.4), 5) E76 + sodium benzoate (J.T.Baker, Deventer, the Netherlands, 0.3 g kg-1 FM, E76 + NaB), 6) E76 + FA0.4 + NaB. Addi- tives FA4 and E76 were applied in the field during harvesting of the grass with the precision chopper. During the filling of the silos, sodium benzoate and FA0.4 were applied as a water solution (20 ml kg-1 FM) into E76-treated herbage, with an equal amount of tap water into UT herbage. In experiment II, herbage was first cut timothy- meadow fescue wilted for 6 h. The treatments con- sisted of 1) No additive (UT), 2) Formic acid 4 l t-1 FM (5 l t-1 AIV2Plus, FA), 3) L. plantarum VTT E-78076 inoculant, freshly cultured, 1 × 106 cfu g-1 (E76), 4) E76 + sodium benzoate 0.3 g kg-1 (E76 + NaB), 5) Commercial inoculant [AIV Biostart, L. plantarum (DSM 4409), BS] and 6) BS + sodium benzoate (BS + NaB). Treatments 2, 3 and 5 were applied to herbage in the field when harvested with the precision chopper while sodium benzoate was added in water solution to treatments 4 and 6 dur- ing silo filling. In both experiments, three replicates for each treatment were prepared as three batches. From the herbages treated in the field, samples were col- lected for each batch (experiment I: UT, FA4, E76 and in experiment II: UT, FA, E76 and BS). The E76 sample in experiment I represents treatments Table 1. Silage additives and post-opening treatments in the experiments I and II. Treatment Silage additives Post opening treatments, g kg-1 fresh matter Experiment I UT Untreated       NaB 0.15, 0.30, 0.45 FA4 Formic acid, 4 l t-1 FM KS 0.15, 0.30, 0.45 E76 L. plantarum VTT E-78076, 1*106 cfu g-1 FM NaB+KS 0.15, 0.30, 0.45 E76+FA0.4 E76+Formic acid, 0.4 l t-1 FM E76+NaB E76+Na-benzoate, 0.3 g kg-1 FM E76+FA0.4+NaB E76+Na-benzoate, 0.3 g kg-1 FM+FA 0.4 l t-1FM Experiment II UT Untreated NaB 0.30 FA Formic acid, 4 l t-1 FM NaB 0.30 E76 L. plantarum VTT E-78076, 1*106 cfu g-1 FM NaB 0.30 E76+NaB E76+Na-benzoate 0.3 g kg-1 FM BS AIVBiostart, L. plantarum DSM 4409 NaB 0.30 BS+NaB AIVBiostart+Na-benzoate 0.3 g kg-1 FM NaB = sodium benzoate, KS = potassium sorbate 188 A G R I C U L T U R A L   A N D   F O O D   S C I E N C E Saarisalo, E. et al. Additives to improve aerobic stability of wilted silage with both FA0.4 and sodium benzoate, and conse- quently, in experiment II E76 and BS represent treatments E76 + NaB and BS + NaB. The silos were covered with a plastic sheet, a plywood lid and weighed down using water con- tainers, resulting in a force of 150 kg m-2, and stored in an unheated barn. The silos were opened in three batches after 145 (±14) or 152 (±14) days of ensiling in experiments I and II, respectively. A layer approximately 15 cm from the top and bot- tom of silage was discarded, and representative samples were collected from the rest for the analy- sis of chemical composition, microbial quality and aerobic stability measurement. Post-opening treatments and aerobic  stability measurement Immediately after opening the silos in experiment I, sodium benzoate, potassium sorbate, (Algol Oy, Finland, KS), or their combination (50:50) at three levels (0.15, 0.30, 0.45 g kg-1 FM) was applied and mixed well to UT, FA4 and LAB silages in water solutions (20 ml kg-1 FM). In experiment II, 0.30 g kg-1 FM sodium benzoate was added to UT, FA, E76 and BS silage. For the aerobic stability measurement 350 g of each silage (intact and post opening treated) was inserted in an open plastic bag into a styrofoam box (volume 1.0 dm3) in duplicates. On the top of the box was a hole (Ø 2 cm) for air to penetrate. A thermistor probe was inserted into the middle of the sample. The boxes were stored at room tem- perature (+21 ± 1oC). The temperature of the si- lages was recorded once a day for 10 days and aerobic stability data are presented as a cumulative difference (sample temperature minus ambient temperature). Chemical and microbiological analysis Chemical and microbial analysis of herbage and silage were carried out as described previously by Saarisalo et al. (2006). Dried samples were ana- lysed for organic matter (OM), nitrogen (N), neu- tral detergent fibre (NDF) and for in vitro organic matter digestibility (OMD) by a modification of the method described by Nousiainen et al. (2003). Fresh samples were stored frozen (–20°C) until analysed for buffering capacity, WSC and soluble N. Oven determined silage DM was corrected with an equation given by Huida (1982). Water extract of silage was measured for pH, WSC, lactic acid, ethanol, ammonia-N and for VFAs (acetic, propi- onic, isobutyric, butyric, isovaleric, valeric and cap- roic acid). Nitrogen and soluble N were measured from fresh samples using the Kjeldahl-method. For quantitative microbial analysis an asepti- cally weighed 10 g sample of grass or silage was suspended in 90 ml of physiological saline con- taining 0.1% peptone and homogenized using a Stomacher (Seward 400, UK) for 30 s at medium power. The samples were analysed for counts of LAB (MRS agar, Oxoid; 30ºC, 3 d, anaerobic in- cubation), aerobic mesophilic bacteria (Plate Count Agar, Difco; 30ºC, 3 d), enterobacteria (Vi- olet Red Bile Glucose (VRBG) Agar, Difco; 37ºC, 18–24 h), clostridia (SFP Agar Base, Difco; 37ºC, 2 d, anaerobic incubation), and yeasts and moulds (Yeast Glucose Chloramphenicol (YGC) Agar, Difco; 25ºC, 5 d). The microbiological analyses were carried out at the Technical Research Centre of Finland (VTT), Espoo. Statistical analysis The silage fermentation and microbial data were tested in both experiments using the SAS GLM procedure with the statistical model: Yi = µ + Ti + ei where Yi is the observation, µ is the overall mean, Ti is the effect of treatment, and ei is the residual error. The sums of squares for treatment effect were further separated by using orthogonal con- trast into single degree of freedom comparisons. In experiment I the following comparisons were used: C1) UT vs. Additives, C2) FA4 vs. E76-treatments, C3) E76 alone vs. E76 + additives, C4) NaB vs. FA0.4, and C5) interaction C3 × C4. C4 and C5 were significant in a very few cases and are there- fore only mentioned in the text. In experiment II, 189 A G R I C U L T U R A L   A N D   F O O D   S C I E N C E Vol. 15 (2006): 185–199. comparisons were: C1) UT vs. Additives, C2) FA vs. Inoculants, C3) E76 vs. BS, C4) Inoculants vs. Inoculants + NaB and C5) interaction C3 × C4. The two last ones were not significant for any of the parameters. The effects of post-opening treatments on cu- mulative temperature in experiment I were tested separately for silages UT, FA4 and E76 and for each day using the following contrasts: C1) No vs. additive, C2) NaB vs. KS, C3) NaB or KS vs. NaB + KS, C4) Linear effect of NaB and KS, C5) Quad- ratic effect of NaB and KS, C6) interaction C2 × C4, C7) interaction C2 × C5, C8) interaction C3 × C4 and C9) interaction C3 × C5. The effects of post-opening treatments on cumulative tempera- ture in experiment II were tested in two ways: Firstly the effect of silage treatment and post-open- ing addition of NaB using contrasts: C1) UT vs. additives, C2) FA vs. LABs, C3) E76 vs. BS, C4) effect of NaB, C5) interaction C1 × C4, C6) inter- action C2 × C4 and C7) interaction C3 × C4. Secondly the effect of time of NaB addition was tested with data from LAB silages: C1) E76 vs. BS, C2) effect of NaB, C3) time of NaB addi- tion (into herbage and post-opening), C4) interac- tion C1 × C2 and C5) interaction C1 × C3. Results The herbages were wilted rapidly under optimal weather conditions so that relatively high DM contents (429 and 344 g kg-1 in experiments I and II, respectively, Table 2) were achieved within six hours. Herbage WSC was on average 134 and 117 g kg-1 DM in experiments I and II, respectively. In experiment I, there were less enterobacteria, LAB, clostridia and yeast in the FA4 treated herbage than in the UT and E76 treated herbage. In ex- periment II, there was a clear decrease in the Table 2. Chemical composition (g kg-1 DM unless otherwise stated) and microbial quality (cfu g-1 FM) of the timothy and meadow fescue grass in experiments I and II. Means of three samples. Experiment I Experiment II UT FA4 E76 UT FA E76 BS Dry matter (g kg-1) 372 474 441 358 323 341 355 Ash 83.2 86.4 80.6 64.8 62.0 64.6 62.5 Neutral detergent fibre 529 498 520 584 566 588 588 Water soluble carbohydrates 128 142 131 112 135 114 108 Nitrogen 22.2 19.2 19.6 24.0 22.8 22.5 22.1 Soluble nitrogen (g kg-1 N) 274 252 256 378 285 331 374 Buffering capacity (mE kg-1DM) 407 420 386 356 434 370 362 In vitro organic matter digestibility 791 804 795 763 771 749 760 Aerobic bacteria 7.8 7.6 7.9 6.8 5.6 6.4 6.5 Enterobacteria 5.5 4.5 4.8 3.8 <1.0 3.7 4.1 Lactic acid bacteria 4.6 2.5 6.5 5.5 3.3 5.7 5.8 Clostridia 1.2 0.5 2.5 1.2 <1.0 1.1 2.2 Yeasts 5.8 4.6 5.9 4.7 4.3 4.7 4.5 Moulds 5.8 5.5 6.0 5.1 2.0 5.2 5.1 UT = untreated, FA4 and FA = Formic acid, 4 l t-1, E76 = L. plantarum VTT E-78076, BS = AIVBiostart, BS = AIVBiostart, L. plantarum DSM 4409. 190 A G R I C U L T U R A L   A N D   F O O D   S C I E N C E Saarisalo, E. et al. Additives to improve aerobic stability of wilted silage number of enterobacteria, LAB and moulds in the FA treated herbage compared to the other treat- ments. Silage fermentation, microbiology and  aerobic stability Experiment I In comparison with the additive treated silages UT silage had higher pH (P < 0.001) and contained more ash (P < 0.05) and less residual WSC (P < 0.001) (Table 3). The ethanol and acetic acid con- centrations, and ammonia-N and soluble N were increased (P < 0.001) in the UT silage compared with the other treatments, while the opposite was observed for the lactic acid content (P < 0.05). The FA4 treatment increased ash content (P < 0.01), pH and WSC (both P < 0.001) compared with the four E76 treatments. There was less lactic and acetic acid (P < 0.001) in the FA4 silage than in the E76 silages. The proportion of ammonia-N was increased in the FA4 silage compared with the E76 silages (P < 0.001) while soluble N was high- er in the E76 than in the FA4 silage (P < 0.05). Table 3. Chemical composition (g kg-1 DM unless otherwise stated), microbial quality (cfu g-1 FM) and aerobic stability of the silages in experiment I. Treatment Statistical significance¤ UT FA4 E76 E76+ NaB E76+ FA0.4 E76+ FA0.4 +NaB SEM C1 C2 C3 Dry matter (g kg(g kg-1) 365 461 444 437 443 436 2.8 Ash 89.3 89.3 85.0 86.1 86.5 86.5 1.00 * ** pH 4.54 4.78 4.06 4.07 4.06 4.05 0.034 *** *** Water soluble carbohydrates 77 207 100 106 105 109 3.1 *** *** o Ethanol 12.7 4.4 4.1 3.7 3.8 3.5 0.37 *** Lactic acid 55.7 6.8 86.7 81.1 82.0 71.4 3.44 * *** o Acetic acid 10.6 5.9 7.2 7.2 7.0 7.2 0.15 *** *** Volatile fatty acids 11.2 6.3 7.6 7.5 7.3 7.5 0.14 *** *** Ammonia-N (g kg-1 N) 66.8 25.9 18.0 18.2 18.9 18.8 0.88 *** *** Soluble N (g kg-1 N) 669 493 558 554 577 567 23.3 *** * Aerobic bacteria 7.8 7.0 5.4 5.4 5.5 5.7 0.33 *** ** Enterobacteria 1.4 1.0 1.0 1.0 1.0 1.0 0.18 * Clostridia 1.2 1.0 1.2 1.1 1.2 1.0 0.14 Lactic acid bacteria 7.7 7.0 5.8 5.7 5.4 5.6 0.32 *** ** Moulds 2.4 2.6 2.1 2.1 1.7 1.7 0.53 Yeasts 4.8 4.8 4.5 4.3 5.4 5.5 0.56 Cumulative Temperature (oC) 3 days 3.5 1.0 1.9 0.1 0.1 0.1 0.90 ** 5 days 16.9 8.7 10.2 2.1 3.3 1.5 3.43 ** o 7 days 32.6 27.6 26.5 7.5 16.2 4.7 5.17 * * ** UT = untreated, FA4 = Formic acid 4 l t-1, E76 = L. plantarum VTT E-78076, FA0.4 = Formic acid 0.4 l t-1, NaB = sodium benzoate 0.3 g kg-1 SEM = standard error of mean ¤Contrasts: C1) UT vs. additives, C2) FA4 vs. all E76, C3) E76 alone vs. E76+NaB and/or FA0.4. Only the significant contrasts are shown. o = P < 0.10, * = P < 0.05, ** = P < 0.01, *** P < 0.001. 191 A G R I C U L T U R A L   A N D   F O O D   S C I E N C E Vol. 15 (2006): 185–199. Within the E76 silages the treatment with E76 alone tended (P < 0.10) to have less WSC (100 vs. 107 g kg-1 DM) and more lactic acid (86.7 vs. 78.2 g kg-1 DM) than the ones in combination with NaB and/or FA0.4. Within the E76 treated silages the E76 + FA0.4 + NaB treated silage contained the lowest lactic acid concentration (interaction P < 0.05). In the UT silage the number of aerobic bacteria and LAB (P < 0.001) were increased compared with the treated silages. Also the enterobacteria count was slightly higher in the UT silage than in other silages (P < 0.05). The LAB and aerobic bac- teria numbers were higher (P < 0.01) in the FA4 than in the E76 silages. Aerobic instability expressed as a cumulative temperature was significantly increased in the UT silage compared to the other treatments after three, five (P < 0.01) and seven days (P < 0.05) exposure to air (Table 3 and Fig. 1a). The cumulative tem- perature of the FA4 treated silage was higher than in the E76 treated silages after seven days (P < 0.05). The cumulative temperature after five days tended to be higher in the E76 silage than in the silages treated with the combination of E76 and NaB and FA0.4 (difference 7.9°C, P < 0.10) and after seven days the difference was increased to 18°C (P < 0.01). Five days after post-opening the number of aerobic bacteria and LAB were lower in additive treated silages as compared with UT (P < 0.05) (Fig. 2). After ten days UT silage had more aerobic bacteria (P < 0.05) and LAB (P < 0.01) and less moulds (P < 0.05) than additive treated silages. At the same time more aerobic bacteria (P < 0.05) and LAB (P < 0.01) were observed in FA than inocu- lated silages. Experiment II In experiment II, ash content tended to be (P < 0.10) higher in the UT silage than in the treated silages (Table 4). FA treatment restricted fermen- tation compared with the inoculated silages result- ing in significantly higher pH, more residual WSC and less lactic acid (all P < 0.001). There was slightly less ethanol (P < 0.10) in the FA silage than in the inoculated silages. Ammonia-N was higher (P < 0.001) in the FA silage than in the in- oculated while the opposite was observed for solu- ble N (P < 0.01). The only differences in the fer- mentation parameters between the E76 and BS si- lages were observed in the concentrations of acetic acid and VFA which were on average 2.17 and 2.60 g kg-1 DM higher (P < 0.001) in the BS than in the E76 silages. The only significant differences in the micro- bial counts which were observed were in the com- parison between FA and the inoculants. In the FA silage there were more enterobacteria (P < 0.01) and LAB and smaller yeasts count (both P < 0.001) than in the inoculated silages. The cumulative temperature of the UT silage was lower than in the other treatments after three days (Table 4 and Fig. 1b). However, the most sig- nificant difference was improved stability of FA Fig. 1. Effect of additives on cumulative temperature of silages in experiments I (a) and II (b). UT = untreated, FA4 and FA = formic acid, E76 = L. plantarum VTT E-78076, BS = AIVBiostart, L. plantarum DSM 4409, NaB = sodi- um benzoate. a) Experiment I 0 5 10 15 20 25 30 35 40 0 1 2 3 4 5 6 7 Days from opening the silo oC UT FA4 E76 E76+FA0.4 E76+NaB E76+FA0.4+NaB b) Experiment II 0 5 10 15 20 25 30 35 40 45 50 0 1 2 3 4 5 6 7 Days from opening the silo oC UT FA E76 E76+NaB BS BS+NaB 192 A G R I C U L T U R A L   A N D   F O O D   S C I E N C E Saarisalo, E. et al. Additives to improve aerobic stability of wilted silage silage compared to the inoculant silages at every time point (P < 0.001). There were no differences within the inoculated silages. Effect of post-opening treatments on  aerobic stability In experiment I, sodium benzoate and potassium sorbate improved the aerobic stability of the UT silage from the second day after opening the silo (P < 0.10) and thereafter more clearly (Fig. 3a). The linear effect on NaB and KS was significant from day four (P < 0.05), and from the fifth day KS tended (P < 0.10) to be more effective than NaB. With FA silage NaB and KS only tended (P < 0.10) to improve aerobic stability during days three and four (Fig. 3b). With E76 silage (Fig. 3c) the effect of additives was significant from the second day (at least P < 0.05) and the linear effect of NaB and KS tended to be significant (P < 0.10) on day six and thereafter significant (P < 0.05). The rest of the contrasts were not significant. Changes in micro- bial quality during aerobic stability measurements are shown in Figure 2. In experiment II, the addition of sodium ben- zoate post opening improved the aerobic stability of the UT, E76 and BS silages by delaying the on- set of warming by one day (Fig. 4). Sodium ben- zoate did not improve aerobic stability of the FA silage. There was a statistically significant differ- ence in the effect of NaB between the inoculants and FA during the first four days (at least P < 0.05, 0 1 2 3 4 5 6 7 8 9 10 0 5 10 Days from opening silo log cfu g-1 Moulds 0 1 2 3 4 5 6 7 8 9 10 0 5 10 Days from opening silo log cfu g-1 Lactic acid bacteria 0 1 2 3 4 5 6 7 8 9 10 0 5 10 Days from opening silo log cfu g-1 Yeasts Aerobic bacteria 0 1 2 3 4 5 6 7 8 9 10 0 5 10 Days from opening silo log cfu g-1 UT FA E76 F E76+A0.4E76+ NaB E76+ FA0.4+ NaB Fig. 2. Microbial quality of the silages at opening and after 5 and 10 days exposure to air in experiment I. UT = untreated, FA = formic acid, E76 = L. plantarum VTT E-78076, NaB = sodium benzoate. 193 A G R I C U L T U R A L   A N D   F O O D   S C I E N C E Vol. 15 (2006): 185–199. and interaction P < 0.10). With the inoculated si- lages the post opening addition of NaB tended to be more effective in improving the stability than NaB addition during the silo filling (time of addi- tion P < 0.05). The rest of the contrasts were not significant. Discussion The compositions of the herbages in experiments I and II were good in terms of ensilability with suf- ficient WSC content (on average 57 and 40 g kg-1 FM in experiments I and II, respectively) and a typical buffering capacity for the grass species used. The lower DM content in the UT herbage in experiment I was unexpected and was probably caused by varying yield and DM concentration of grass on the field. Formic acid 4 l t-1 and the inoculants were ap- plied during harvesting resulting in immediate pH drop on herbage as in typical ensiling. NaB and FA0.4 were applied during filling the silos. These additives are expected to have minor effects on composition of grass between harvesting and en- siling. The WSC content in the herbage, sampled Table 4. Chemical composition (g kg-1 DM unless otherwise stated), microbial quality (cfu g-1 FM) and aerobic stability of the silages in experiment II. Treatment SEM Statistical significance¤ UT FA E76 E76+ NaB BS BS+ NaB C1 C2 C3 Dry matter, g kg-1 347 313 329 329 344 343 2.7 *** *** *** Ash 67.5 65.7 66.0 66.3 65.1 66.2 0.71 o pH 4.10 4.34 3.92 3.92 3.92 3.93 0.034 * *** Water soluble carbohydrates 40 138 36 42 41 44 2.6 *** *** Ethanol 9.7 7.9 10.5 10.2 8.2 8.0 0.67 o Lactic acid 86 11 101 99 100 97 1.6 * *** Acetic acid 9.7 8.0 6.3 7.2 8.9 9.0 0.47 ** *** Volatile fatty acids 10.0 8.3 6.5 7.4 9.2 9.9 0.60 * *** Ammonia-N, g kg-1 N 51.9 28.4 22.7 22.6 22.9 23.1 1.14 *** *** Soluble N, g kg-1 N 683 590 648 633 655 650 14.9 * ** Neutral detergent fibre 571 566 573 573 574 574 2.9 * Aerobic bacteria 6.7 5.9 6.2 6.5 6.4 6.2 0.22 o Enterobacteria 0.5 1.7 0.5 0.5 0.5 0.5 0.27 ** Clostridia 0.5 1.0 0.5 0.5 0.5 1.0 0.15 * Lactic acid bacteria 6.0 7.2 6.0 6.2 6.1 6.0 0.22 *** Moulds 1.4 1.7 1.1 1.0 1.2 1.7 0.24 Yeasts 5.9 4.2 6.1 6.4 6.1 6.0 0.32 *** Cumulative Temperature oC 3 days 14.3 0.0 23.1 23.9 22.8 21.9 1.70 * *** 5 days 29.2 3.7 34.7 37.7 33.7 35.2 2.65 *** 7 days 39.6 21.2 43.8 47.6 41.9 45.3 3.39 *** UT = untreated, FA = Formic acid 4 l t-1, E76 = L. plantarum VTT E-78076, BS = AIVBiostart, NaB = sodium benzoateBS = AIVBiostart, NaB = sodium benzoateNaB = sodium benzoate= sodium benzoatesodium benzoate 0.3 g kg-1 FM, SEM = Standard error of mean, SEM = Standard error of mean ¤ Contrasts: C1) UT vs. Additives; C2) FA vs. Inoculats; C3) E76 vs. BS. Only the significant contrasts are shown. o = P < 0.10, * = P < 0.05, ** = P < 0.01, *** = P < 0.001 194 A G R I C U L T U R A L   A N D   F O O D   S C I E N C E Saarisalo, E. et al. Additives to improve aerobic stability of wilted silage content in the FA herbage is inhibition of respira- tion between the time of additive application and freezing of the samples. Effect of treatments on silage  fermentation It is well documented that the fermentation quality of low DM grass silage can be improved by FA based silage additives (Waldo 1977, Jaakkola et al. 1991, Kung et al. 2003, Jaakkola et al. 2006) while with inoculants results have been more variable (Weinberg and Muck 1996, Pobednov et al. 1997, Kung et al. 2003). With the low DM grass silages the positive effects of inoculants on silage fermen- tation depend on the adequate amount of WSC (Anderson et al. 1989) and often no effect com- pared to untreated has been observed with low ini- tial WSC (e.g. Keady and Murphy 1997, Yan et al. 1998). Fewer studies have been conducted to com- pare additives with rapid wilted, high DM (≥330 g kg-1) grass silages. In the present experiments, all the silages were of good quality and none contained excessive con- centrations of acetic, butyric or other VFAs, or 0 5 10 15 20 25 30 35 40 45 50 0 1 2 3 4 5 6 7 Days from opening the silo oC UT FA E76 BS UT_NaB FA_NaB E76_NaB BS_NaB Fig. 4. Effect of post opening addition of sodium benzoate (NaB) on aerobic stability of silages in experiment II. UT = untreated, FA = formic acid, E76 = L. plantarum VTT E-78076, BS = AIVBiostart, L. plantarum DSM 4409 c) E76 0 5 10 15 20 25 30 35 0 1 2 3 4 5 6 7 Days from opening the silo oC a) Untreated 0 5 10 15 20 25 30 35 0 1 2 3 4 5 6 7 Days from opening the silo oC No NaB_15 NaB_30 NaB_45 KS_15 KS_30 KS_45 NaBKS_15 NaBKS_30 NaBKS_45 b) Formic acid 0 5 10 15 20 25 30 35 0 1 2 3 4 5 6 7 Days from opening the silo oC Fig. 3. Effects of post-opening treatments with sodium benzoate (NaB) and potassium sorbate (KS) at three ap- plication rates (0.15, 0.30 and 0.45 g kg-1 FM) on cumula- tive temperature of untreated (a), formic acid treated (b) and inoculant (E76) treated (c) silages in experiment I. during filling the silos, 2–3 hours after additive ap- plication, was higher in the FA treated than in the untreated or inoculated herbage agreeing with the observations of Keady and Murphy (1997). Re- spectively, the NDF content was smaller in the FA herbage suggesting WSC releasing acid hydroly- sis. Another possible reason for the highest WSC 195 A G R I C U L T U R A L   A N D   F O O D   S C I E N C E Vol. 15 (2006): 185–199. ethanol. However, all the additives improved the fermentation quality compared with the UT silage, especially in terms of the ammonia-N, which was 45 and 28 g kg-1 N higher in the UT than in the ad- ditive treated silages in experiments I and II, re- spectively. In experiment I, the difference in the fermentation quality of the UT and the additive treated silages was greater than in experiment II. This was probably caused by the lower DM con- tent in the UT than in other silages in experiment I, resulting in more extensive fermentation of the UT silage. Despite the reasonable high DM content of si- lage in both the experiments, there were noticeable differences in the fermentation type between the FA and inoculant treatments, though both were equally effective in decreasing ammonia produc- tion. All the treatments resulted in high concentra- tions of residual WSC, which resulted mainly from the moderate formation of fermentation acids. In the FA silages, formic acid, together with the high DM, restricted production of lactic and other fer- mentation acids, resulting in a pH 0.7 and 0.4 higher than in the inoculated silages in experi- ments I and II, respectively. The LAB inoculants enhanced lactic acid production, resulting in lower pH, which improves hygienic quality regarding potential silage transmitted pathogens like Liste- ria, coliforms, Bacillus and Clostridia (Jonsson et al. 1990, McDonald et al. 1991). The capability of inoculants to improve silage fermentation at high dry matter was also observed by Jonsson et al. (1990) and Driehuis et al. (1997). Regarding the novel LAB inoculant, the results agree with the previous laboratory scale experiment in which E76 resulted in fast lactic acid production, a drop in pH and restricted ammonia production (Saarisalo et al. 2006). E76 was equally effective as the com- mercial inoculant strain in BS (L. plantarum DSM 4409). Effect of treatments on aerobic stability  Aerobic deterioration decreases the nutritive value of silage, impairs the hygienic quality and, in addi- tion to animals, may cause health risks to people working with animals and even to consumers via milk as reviewed by Woolford (1990), Lindgren (1991) and Wilkinson (1999). The majority of re- search on the factors affecting aerobic stability has focused on maize and small grain cereal silages (Driehuis et al. 1999, Weinberg et al. 1999, Uriarte and Bolsen 2001, Danner et al. 2003) indicating that the aerobic instability has been a greater prob- lem with those materials having a higher DM con- tent and a coarser physical structure than grass. It is possible that the fermentation pattern, microbial flora and course of aerobic deterioration in maize and small grain cereal silages are somewhat differ- ent from wilted grass silage. In both of our experi- ments, aerobic stability of grass silages was rela- tively poor, since increased temperature was ob- served already from day one (experiment I) or day three (experiment II) after opening the silos. The complexity of the factors affecting aerobic stability of silage was highlighted and some of the results disagree with the previous observations on the re- lationship between the chemical composition and the aerobic stability. A high DM content has been mentioned as a factor impairing the stability of grass silage (Yan et al. 1998, Wyss 1999). However, the UT silage had the lowest stability in experiment I despite having the lowest DM content. In addition, the silages in experiment I with an average DM of 431 g kg-1 were more stable than the silages in experiment II with an average DM of 327 g kg-1. These observa- tions suggest that DM content of silage alone does not predict the susceptibility to aerobic deteriora- tion well. Another major factor considered to affect aero- bic stability is the fermentation type of silage. In particular, extensive heterolactic fermentation and butyric and acetic acid have improved stability (McDonald et al. 1991, Danner et al. 2003). In our experiments, the UT silage with the highest acetic acid concentration was less stable than the FA treated restrictively fermented silage. Furthermore, the inconsistency between the fermentation type and aerobic stability was emphasised by the results with the LAB inoculants. Despite similar homo- lactic fermentation in experiments I and II, the in- oculants produced dissimilar effects on aerobic 196 A G R I C U L T U R A L   A N D   F O O D   S C I E N C E Saarisalo, E. et al. Additives to improve aerobic stability of wilted silage stability. In experiment I, E76 improved aerobic stability compared to UT, while in experiment II, both inoculants produced less stable silage than UT and especially FA. Variable effects of homo- lactic inoculants are not rare in the literature (McDonald et al. 1991, Uriarte and Bolsen 2001). The application rate of 4 l FA t-1 grass, as rec- ommended in Finland, resulted in good aerobic stability, especially in experiment II, despite re- stricted fermentation, high WSC content and high pH, which have been considered as risk factors for aerobic stability. Another noteworthy observation was that the yeast count was not increased in the FA silages as often observed (McDonald et al. 1991, Uriarte and Bolsen 2001). The varying ef- fect of FA on aerobic stability is not uncommon and possibly due to different microorganisms re- sponsible for aerobic deterioration (Kung et al. 2003). An explanation for our good results with rather high rate of FA might be the effective con- solidation of the acid treated herbage during silo filling, leading to increased storage density and thereby decreased air infiltration into the silage. A way to decrease both aerobic instability and improve silage fermentation is to combine homo- lactic LAB inoculant with chemical additives. Re- striction of fermentation by wilting and LAB in- oculation with chemical preservatives can be re- garded as an application of the hurdle technology (Leistner and Gorris 1995). For example, combi- nation of an inoculant and sodium formate was studied by Weissbach et al. (1991). Sodium ben- zoate and potassium sorbate are weak-acid pre- servatives effective against yeasts and moulds (Woolford 1975) and commonly used as food pre- servatives. Their potential as antimicrobial agent depends on the proportion of undissociated acids which pass through the cell membranes and liber- ate protons, thus acidifying the cytoplasm and pre- venting growth of microbes (Lambert and Strat- ford 1999). In addition, the effect depends on pH as the proportion of the undissociated acids in- creases with declining pH. In the present experi- ments, two different methods to add chemical agents were used, either application during ensil- ing or post-opening the silos. Using the latter method it is possible to study the potential of these additives with more treatments than it would have been practically possible within an ensiling experi- ment. A similar approach has also been used by Weinberg et al. (1993) in a study on effects of ace- tic and propionic acid. The chemical additives FA0.4 (0.4 l t-1 FM) in experiment I, and NaB (0.3 g kg-1 FM) in both ex- periments applied to the inoculated silages during ensiling did not have any adverse effect on the ef- ficiency of inoculants since silage fermentation was not affected. However, FA0.4 applied as free acid had to be added separately since in a previous laboratory experiment LAB was eliminated when applied mixed with the same amount of FA as in the current experiment. In experiment I, FA0.4 and/or NaB improved aerobic stability of the E76 silage even though the yeast count was smaller in E76 without the chemi- cal additives than in E76 + FA04 and E76 + FA0.4 + NaB silages. There were no other clear differ- ences in the microbial counts that could explain this improved aerobic stability of inoculated si- lages. On the other hand, microbial changes during exposure to air (Fig. 2) clearly reflected aerobic stability as the number of moulds and yeasts did not increase in the E76 + chemical additive silages in which the smallest temperature increase was ob- served after five days. This supports the hypothesis that the chemical additives in combination with an inoculant restrict microbes responsible for aerobic deterioration, which is in line with the results of Weissbach et al. (1991) and Rammer et al. (1999). In experiment II, NaB added at the same rate as in experiment I had only a minor effect on aerobic stability. Rammer et al. (1999) reported prolonged storage stability with 0.20 and 0.40 g kg-1 NaB in combination with an inoculant applied to grass and grass-legume herbage. However, fermentation pa- rameters were not reported. According to Lingvall and Lättemäe (1999), 0.8 g kg-1 NaB or 0.69 NaB + 0.21 sodium propionate was enough to control aerobic stability of baled grass silage (DM 330– 350 g kg-1, pH 4.3–4.4). Kleinschmit et al. (2005) found that 1 g kg-1 of NaB and also 1 g kg-1 maize silage of KS plus EDTA improved stability. Re- garding the variability in reasons for onset of dete- rioration, it is clear that the minimum effective ap- 197 A G R I C U L T U R A L   A N D   F O O D   S C I E N C E Vol. 15 (2006): 185–199. plication rate also varies. Sodium benzoate and potassium sorbate applied post-opening were more effective in improving the stability of inoculated silages than FA silages, which can be explained by the clear difference in silage pH affecting the pro- portion of dissociated acids. Within the experiments the relationship be- tween the microbial quality and aerobic stability was not straightforward. However, the poorer aer- obic stability of the inoculated silages in experi- ment II could be explained by the yeast count which was a hundred times higher (log 4 vs. log 6) in experiment II than in experiment I. The number of aerobic bacteria was also ten times higher in ex- periment II. In the present study, microbial popula- tion was examined quantitatively, only at a general level. More detailed characterisation of the yeast population would be needed for explaining the role of lactate fermenting yeasts in the aerobic spoilage process. Yeast population differences in first and second cut silages may also play an important role. Antimicrobial properties of E76 might be a factor in ensuring rapid proliferation at early phas- es of silage fermentation and consequently a good fermentation pattern. However, as observed in the previous study (Saarisalo et al. 2006), LAB num- bers in final silages were smaller in inoculated than in the restrictively fermented or in UT silage, prob- ably due to low pH and autolysis. In conclusion, the novel LAB inoculant was equally effective in improving fermentation quali- ty, and especially in reducing ammonia formation in grass silage, compared with the commercial LAB inoculant. However, the aerobic stability of inoculant-treated silages was poorer than that of formic acid treated or untreated silages in one of the experiments. The results suggest that the anti- microbial properties of E76 were not effective enough to improve aerobic instability. One option to overcome this problem is to use chemical addi- tives in combination with the inoculants. The re- sults suggest that the minimum effective applica- tion rate of sodium benzoate varies. References Anderson, R., Gracey, H.I., Kennedy, S.J., Unsworth, E.F. & Steen, R.W.J. 1989. 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Esikuivatus vähentää säilörehun käymisen laatuongel- mia, mutta voi johtaa siihen, että rehut lämpenevät her- kemmin siilon avaamisen jälkeen. Kahdessa kokeessa tutkittiin uuden maitohappobakteerikannan, Lactobacil- lus plantarum VTT E-78076 (E76), tehokkuutta säilöre- hun biologisena säilöntävalmisteena. Säilörehujen käy- mislaadun lisäksi tutkittiin rehujen jälkipilaantumis- herkkyyttä eli aerobista stabiilisuutta ja mahdollisuutta parantaa sitä yhdistämällä biologinen ja kemiallinen säi- löntävalmiste. Säilörehujen raaka-aine oli esikuivattua timotei-nur- minataa, jonka kuiva-ainepitoisuus oli ensimmäisessä kokeessa 429 g/kg ja toisessa 344 g/kg. Säilöntäainekä- sittelyjä oli kuusi. Kokeen I käsittelyt olivat E76 yksin (106 pmy/g), E76 yhdessä natriumbentsoaatin (0,30 g/kg ruohoa) tai lievän muurahaishapon (0,40 g/kg ruohoa) sekä näiden yhdistelmän kanssa. Kokeen II käsittelyt olivat E76 ja kaupallinen biologinen valmiste (AIVBio- profit, L. plantarum DSM 4409, 106 pmy/g) sekä yksin että yhdessä natriumbentsoaatin (0,30 g/kg ruohoa) kanssa. Kontrolleina molemmissa kokeissa olivat käsit- telemätön eli painorehu ja muurahaishappopohjainen säilöntäaine (AIV2Plus 5 l tonnille). Aerobisen stabiili- suuden parantamista tutkittiin myös lisäämällä natrium- bentsoaattia ja kaliumsorbaattia rehuihin siilojen avaa- misen jälkeen. Uusi maitohappobakteerikanta tuotti painorehua pa- remman ja kaupalliseen valmisteeseen verrattuna yhtä hyvän käymislaadun. Maitohappobakteerirehujen aero- binen stabiilisuus oli kuitenkin molemmissa kokeissa huonompi kuin muurahaishapporehun, ja toisessa ko- keessa myös painorehu oli stabiilimpaa. Aerobista sta- biilisuutta voitiin parantaa yhdistämällä maitohappo- bakteeri ja kemiallinen säilöntäaine. Woolford, �.K. 1990. The detrimental effects of air on si� lage. Journal of Applied Bacteriology 68: 101–116. Wyss, U. 1999. In��uence of �re�wilting degree on aerobic stability of grass silages. In: Pauly, T. (ed.). Proceed- ings of the 12th International Silage Conference, 5–7 July, U��sala, Sweden. �. 284–285. Yan, T., Patterson, �.C., Gordon, F.J. & Kil�atrick, �.J. 1998. Effects of bacterial inoculation of unwilted and wilted grass silages. 1. Rumen microbial activity, silage nutri� ent digestibility and digestibility. Journal of Agricultural Science, Cambridge 131: 103–112. SELOSTUS Maitohappobakteerien, muurahaishapon, natriumbentsoaatin ja kaliumsorbaatin vaikutus  esikuivatun säilörehun käymislaatuun ja aerobiseen stabiilisuuteen Eeva Saarisalo, Taina Jalava, Eija Skyttä, Auli Haikara ja Seija Jaakkola Maa- ja elintarviketalouden tutkimuskeskus ja VTT Effect of lactic acid bacteria inoculants, formic acid, potassium sorbate and sodium benzoate onfermentation quality and aerobic stability ofwilted grass silage Introduction Material and methods Results Discussion References SELOSTUS