The effect of temperature on the ensiling process of corn and wheat

Transcription

1 Journal of Applied Microbiology 2001, 90, 561±566 The effect of temperature on the ensiling process of corn and wheat Z.G. Weinberg 1, G. Szakacs 2, G. Ashbell 1 and Y. Hen 1 1 Forage Preservation and By-Products Research Unit, The Volcani Center, Bet Dagan, Israel, and 2 Department of Agricultural Chemical Technology, Technical University of Budapest, Hungary 622/11/00: received 3 November 2000 and accepted 19 December 2000 Z. G. W E I N B E R G, G. S Z A K A C S, G. A S H B E L L A N D Y. H E N Aims: The purpose of this work was to study the effect of temperature on the ensiling process and aerobic stability of corn and wheat silages. Methods and Results: The crops were ensiled in 1á5 l anaerobic jars, with and without an inoculant, at room or elevated temperatures (37±41 C). After two months of ensiling, the silages were subjected to an aerobic stability test at room and elevated (33 C) temperature. The results indicate that ensiling at elevated temperatures resulted in higher ph values, less lactic acid and higher losses. The silages which were stored at elevated temperatures were more susceptible to aerobic spoilage than those stored at room temperature, especially when the test was performed at elevated temperature. Conclusions: High temperatures are detrimental to both the ensiling process and the aerobic stability of silages. Signi cance and Impact of the Study: The ndings of the current study suggest that in a warm climate, special care should be taken during silage making and storage in order to avoid heating as much as possible. In addition, in a warm climate, silages are more susceptible to aerobic deterioration and therefore, special care should be taken during unloading. INTRODUCTION Ensiling is a conservation method for moist crops. It is based on natural fermentation under anaerobic conditions, whereby epiphytic lactic acid bacteria (LAB) convert water-soluble carbohydrates (WSC) into organic acids, mainly lactic acid (LA). As a result, the ph decreases and the forage is preserved. Air is detrimental to the ensiling process as it permits the activity of aerobic spoilage micro-organisms (Woolford 1990). In commercial silages, during the initial stages of fermentation, when air is still present between the plant particles, the temperature might rise to 40 C and above because of continuing plant respiration and aerobic microbial activity. In a warm climate, the high temperature may persist in the silage for several months (Jiang et al. 1988; Ashbell and Weinberg 1992; Weinberg and Ashbell 1994). Silage is often exposed to air when it is in the silo because sealing is not hermetic, and spoilage might have Correspondence to: Dr Z.G. Weinberg, Forage Preservation and By-Products Research Unit, The Volcani Center, Bet Dagan, Israel ( zgw@volcani.agri.gov.il). already started at this stage (Honig 1991; Weinberg and Ashbell 1994). During unloading, the silage might be fully exposed to air resulting in an increase in temperature and spoilage (aerobic deterioration). Micro-organisms can be categorized, according to their ability to grow at low, moderate or high temperatures, as psychrophilic, mesophilic and thermophilic. The temperature ranges of LAB vary and different LAB have different temperature optima (McDonald et al. 1991). Since ensiling is a fermentation process in which LAB play an important role, it might be strongly affected by the temperature within the silage. An early study by McDonald et al. (1966) showed that high temperature (42 C) during ensiling of moist grass resulted in a shift from lactic to a clostridial type of fermentation, with increased losses. Muck and Dickerson (1988) showed that increasing the storage temperature from 15 to 35 C enhanced proteolysis and resulted in elevated ammonia concentrations in alfalfa silages. In addition, the success of bacterial inoculants, which are sometimes used to enhance the ensiling fermentation, might also depend on the temperature. For example, a previous study (Weinberg et al. ã 2001 The Society for Applied Microbiology

2 562 Z.G. WEINBERG ET AL. 1998) revealed that the activities of Lactobacillus plantarum and Lact. amylovorus added to wheat silage differed in activities at 25 and 40 C. The purpose of the current work was to extend the study of the effect of temperature on the ensiling fermentation, with or without LAB inoculant, and on the aerobic stability of corn and wheat silages. MATERIALS AND METHODS Ensiling of corn Chopped, fresh, whole plant corn was bought from a commercial feeding centre and ensiled in 72 1á5 l glass jars (Weck, Wher-Oftlingen, Germany) equipped with a lid that enabled gas release only. Each jar was lled with about 550 g (wet weight) chopped corn without a headspace. Thus, the packing density obtained was 366 kg m ±3, which is about 65% of the density in commercial silos in Israel (Weinberg et al. 1993). The treatments comprised a control (no additives) and a commercial LAB inoculant (H/M F, Medipharm USA, Des Moines, IA, USA), containing a minimal count of colony-forming units (cfu) g ±1 powder of Pediococcus acidilactici, Lactobacillus plantarum and Enterococcus faecium (manufacturer's statement). The number of cfu was validated by enumerating the inoculant LAB on Rogosa agar. The inoculant was applied by suspending 500 mg H/M F in 50 ml tap water which was sprayed on 25 kg of the chopped crop, spread over 1 4m, followed by thorough mixing. Thus, 10 5 cfu g ±1 were applied. The jars were stored under the following temperature regimes: (i) constant room temperature (28 1 C); (ii) constant high temperature (37 C); (iii) variable temperature, i.e. the initial 2 weeks at 41 C, the following 2 weeks at 37 C and the nal 4 weeks at room temperature (27±28 C). Three jars per treatment from every temperature were sampled on days 2, 5, 22 and 63 for ph measurement and LAB enumeration. The nal silages (day 63) were subjected to full analysis. At the end of the experiment the silages were also subjected to an aerobic stability test at room temperature (27 C), lasting 6 days, in a system developed by Ashbell et al. (1991). The system is constructed from two parts of recycled soft drink bottles (polyethylene terephtahlate). The upper part (1 litre) is lled with about 250 g (wet weight) loosely-packed silage and the lower part, with 100 ml 20% KOH. Gas is exchanged through 1 cm holes in the upper part. Carbon dioxide produced during aerobic exposure is absorbed in the base and determined by titration with 1N HCl. In addition, change in ph, yeast and mould counts, and visual appraisal, also serve as indicators for aerobic spoilage. Ensiling of wheat A similar experiment with wheat was performed in spring, with the following changes. The temperature regimes comprised only room temperature (24 1 C) and constant high temperature (41 C). The sampling dates were 1, 3, 10 and 60. The aerobic stability test of the silages from both temperature treatments was performed at room temperature (26 C) and at 33 C, and lasted 5 days. Analytical procedures Dry matter was determined by oven-drying for 48 h at 60 C. Water-soluble carbohydrates were determined by the phenol sulphuric acid method, according to Dubois et al. (1956). Lactic acid was determined by a spectrophotometric method, according to Barker and Summerson (1941). Volatile fatty acids were determined using a gas chromatograph with a semi-capillary FFAP column (Hewlett Packard, Waldbronn, Germany) over a temperature range of 45 to 230 C. Acid detergent bre (ADF) and acid detergent insoluble nitrogen (ADIN) were determined according to van Soest (1982). Ammonia was determined in control silages by extraction of 40 g frozen samples with 360 ml distilled water for 3 min in a Stomacher blender; 100 ml of the extract were used for distillation in the Kjeldahl unit without a digestion step. The microbiological evaluation included the enumeration of lactobacilli (on pour plate Rogosa agar; Oxoid), and yeasts and moulds (on spread plate malt extract agar acidi ed with lactic acid to ph 4á0). All plates were incubated for 3 days at 30 C. This analysis was performed on a single representative sample. Statistical analysis of the silage chemical results included a one-way analysis of variance and Duncan's multiple range test performed with the Statistical Analysis System (SAS, Cary, NC, USA). RESULTS Corn silages The fresh corn contained 394, 32 and 60 g kg ±1 DM, WSC and crude protein, respectively, and the ph was 5á9. The log numbers of cfu g ±1 DM of LAB, yeasts and moulds in the fresh material were 7á4, 7á4 and 5á7, respectively. The ph changes of the various treatments are given in Fig. 1. The ph values of the inoculated silages were similar to those of the uninoculated silages at the same temperature throughout the ensiling period. In the early stages of fermentation (until day 5), the ph values of the silages stored at different temperatures did not differ markedly; in later stages of fermentation (days 22 and 63) and after

3 EFFECT OF TEMPERATURE ON ENSILING 563 exposure (Table 3) and therefore, no temperature or inoculant effect could be observed. Fig. 1 ph change in corn silages. (d), Room temperature, 28 C; (j), high temperature, 37 C; (m), changing temperature 41 to 37 to 28 C. A6, after aerobic exposure for 6 days exposure to air, the ph values of the silages stored at room temperature were signi cantly lower than those stored at the higher temperatures. Table 1 gives the chemical analyses of the nal corn silages. In the silages stored at the high temperature, less lactic acid was produced, which is consistent with their nal higher ph values; in these silages, higher losses were observed. No residual WSC were found in any of the silages. More ammonia-n was found in the control silages which were stored at elevated temperatures. Within a temperature regime there were only slight differences between the control and inoculated silages and these were not statistically signi cant. Statistical analysis revealed signi cant (P < 0á05) temperature effects on nal ph, lactic acid and weight losses; at elevated temperatures, higher ph, less lactic acid and higher losses were obtained. Dry matter was highest in silages stored at 37 C and lowest in those with changing temperature regime. No signi cant inoculant effects were observed for any of the measured parameters. Signi cant temperature inoculant interactions were obtained only for ph. The numbers of LAB in the control corn silages under the high temperature regime were generally lower than those under the room or variable temperature regimes (Table 2). The number of yeasts in all the nal silages was low (log cfu < 2á0). All corn silages were stable during aerobic Wheat silages The fresh wheat contained 420, 127 and 63 g kg ±1 DM, WSC and crude protein, respectively, and the ph was 6á0. The log numbers of cfu g ±1 DM of LAB, yeasts and moulds in the fresh material were 4á1, 4á4 and 4á0, respectively. The ph changes of the various wheat silages are given in Fig. 2. After 1 day of fermentation, the ph values of the silages stored at 41 C were lower than those of the respective silages which were stored at 24 C. From day 3 onwards, throughout the ensiling period, the ph values of the warmer silages were higher than the values of those stored at room temperature; during this period, no differences were noticed between the control and inoculated silages. Table 4 gives the chemical analyses of the nal wheat silages. As with the corn silages, signi cantly (P < 0á05) less lactic acid was produced, and higher ph values were obtained, in the wheat silages which were stored at the higher temperature. Neither inoculant nor temperature had a signi cant (P < 0á05) effect on losses. Residual WSC were signi cantly (P < 0á05) higher at 41 C, which, along with the lower lactic acid content, indicate less intensive fermentation at this temperature. No differences in ammonia-n were observed between wheat silages stored at room and high temperatures. Treatment with the inoculant had signi cant effects on nal ph, ethanol and acetic acid contents; the inoculated silages had higher ph values, higher ethanol and lower acetic acid contents. No signi cant temperature inoculant interactions were obtained for any of the measured silage parameters. The numbers of LAB of the wheat silages are given in Table 5. On day 3 after ensiling, the control silages from 41 C had lower numbers of LAB than those from 24 C; the inoculated silages from 41 C had somewhat more LAB than those from 24 C. On day 10, the number of LAB in the silages stored at 41 C was lower than those stored at 24 C, regardless of inoculation. By day 60, the number of LAB Table 1 Chemical analyses of the nal corn silages. (g kg ±1 in DM S.E.) Temperature Treatment Dry matter ph Lactic acid Ethanol Acetic acid NH 3 -N/TN % Weight loss 28 C Control á á6 0á2 16á3 2á8 0á11 0á01 0á6 0á1 Inoculant á á4 0á8 10á9 1á9 0á5 0á0 37 C Control á á7 0á1 16á8 1á1 0á15 0á01 1á0 0á0 Inoculant á á8 0á4 14á4 2á0 1á0 0á0 41±37±28 C Control á á5 0á4 14á0 3á7 0á14 0á01 0á9 0á0 Inoculant á á5 0á4 14á5 1á0 0á8 0á0 Signi cant (P < 0á05) temperature effects were obtained for ph, lactic acid and weight losses.

4 564 Z.G. WEINBERG ET AL. Table 2 The number of LAB during ensiling of corn (log cfu g )1 DM) Temperature Treatment Day 2 Day 5 Day 22 Day C Control 9á6 9á6 8á7 7á9 Inoculant 9á7 9á7 9á4 7á8 37 C Control 8á3 8á9 7á0 6á7 Inoculant 9á6 9á4 7á3 7á1 41±37±28 C Control 9á4 8á7 6á5 8á5 Inoculant 9á5 8á8 7á0 8á5 Table 3 Results of the aerobic stability test of the corn silages after 6 days of exposure. Yeasts and moulds are given as log cfu g )1.No CO 2 was produced in these tests Temperature Treatment ph Yeasts Moulds 28 C Control 4á1 0á1 <2á0 3á9 Inoculant 4á0 0á0 4á8 5á4 37 C Control 4á4 0á1 <2á0 2á1 Inoculant 4á4 0á0 <2á0 <2á0 41±37±28 C Control 4á5 0á0 <2á0 4á6 Inoculant 4á5 0á0 <2á0 3á5 had decreased in all silages, and more so at 41 C. Yeasts were found in substantial numbers only in the inoculated silages which were stored at 24 C (log cfu g ±1 DM ˆ 4á8); these silages also had some mould counts (log cfu g ±1 DM ˆ 2á4). The results of the aerobic stability test are given in Table 6. Samples which were exposed to air at elevated temperature (33 C) tended to deteriorate more than those which were exposed to air at room temperature (26 C). This was indicated by the CO 2 production and large numbers of yeasts and moulds. In this experiment, aerobic deterioration was not accompanied by change in ph, and the ph values after exposure to air re ect the ph values of the silages before exposure to air. This was probably due to the high levels of residual WSC that the yeasts could use instead of lactic acid, which did not result in a change in ph. The statistical analyses of the aerobic stability test revealed highly signi cant effects on CO 2 production of both the ensiling (P < 0á01) and aerobic exposure (P < 0á01) temperatures; inoculation did not have a statistical signi cant effect on CO 2 production, but more samples of the inoculated silages deteriorated upon exposure to air than of the control silages. Table 7 gives the results of the ADF and ADIN analyses. ADF values of the nal silages were higher than those of the fresh material, in both the corn and the wheat, re ecting loss of organic matter during ensiling. However, no marked differences between temperature treatments were observed in either ADF or ADIN. Fig. 2 ph change in wheat silages. (d), C-24, control at 24 C; (n), C-41, control at 41 C; (m), I-24, inoculated at 24 C; (s), I-41, inoculated at 41 C DISCUSSION Ensiling is based on fermentation, which involves microbiological and enzymatic activity. Therefore, it is expected to be strongly in uenced by temperature. In most of the literature describing ensiling experiments, there is no reference to the temperature at which the ensiling was carried out. In farm-scale ensiling in a warm climate, however, the temperature within the silage often increases to around 40 C, due to plant respiration and microbiological Table 4 Chemical analyses of the nal wheat silages. (g kg )1 in DM S.E.) Temperature Treatment Dry matter ph* Residual WSC Lactic acid Ethanol Acetic acid NH 3 -N/TN % Weight loss 24 C Control á á12 0á8 0á1 Inoculant á á0 0á2 41 C Control á á12 0á8 0á1 Inoculant á á7 0á2 *S.E. for ph were less than 0á05. Signi cant (P < 0á05) temperature effects were obtained for ph, lactic and acetic acids, and residual WSC; Signi cant (P < 0á05) inoculant effects were obtained for ph, acetic acid and ethanol.

5 EFFECT OF TEMPERATURE ON ENSILING 565 Table 5 The number of LAB during ensiling of wheat (log cfu g )1 DM) Temperature Treatment Day 1 Day 3 Day 10 Day C Control 9á0 9á7 9á3 6á3 Inoculant 9á3 7á7 9á5 6á3 41 C Control 9á1 8á3 7á8 4á8 Inoculant 8á9 8á5 7á5 4á6 activity, and it remains high for many months (Jiang et al. 1988; Ashbell and Weinberg 1992; Weinberg and Ashbell 1994). Air may penetrate the silage during storage and when the silage is unloaded for feeding, it is fully exposed to air and can warm up again because of aerobic microbial activity. This could result in losses and affect the quality of the silage, and might have implications for its aerobic stability and the success of silage inoculants. Therefore, it was decided to simulate in the laboratory the eld conditions which exist on the farm. In the corn experiment, a high temperature regime was included which was gradually lowered, as happens frequently on the farm; since no major differences were observed between this treatment and the constant high temperature treatment (37 C), only room and constant high temperature (41 C) treatments were included in the wheat experiment. McDonald et al. (1966) found that in moist grass silages which were warmed to 42 C, losses were higher than in cool silages, and the fermentation changed from lactic to clostridial. In the warm silages, less lactic acid was produced and the ph remained high compared with silages fermented at 20 C. In wilted grass silage (250 g kg ±1 DM), the temperature effect was less pronounced. The present results with the much drier corn and wheat silages (400 g kg ±1 DM) are consistent with the results of McDonald et al. (1966); the silages stored at high temperatures had higher ph values, less lactic acid was produced and their losses were larger than in those stored at the lower temperature. In the present study, the inoculant effect on ensiling was minor under all temperature regimes, as occurs sometimes with easy-to-ensile crops. In another study (Weinberg et al. 1998), wheat silages which were inoculated with Lact. plantarum had higher LAB counts at 25 C than at 41 C, whereas in those inoculated with Lact. amylovorus the opposite held true. These results indicate that the success of an inoculant for silage might depend on the ability of the LAB comprising it to withstand warm conditions. Aerobic deterioration of silage is a complex process which depends on many factors. Usually it is initiated by aerobic yeasts which can use either residual WSC or lactic acid for their metabolism. Aerobic deterioration usually results in production of CO 2 and consequent DM losses. It is assumed that when high levels of residual WSC are available for the aerobic yeasts, no change in ph will occur during aerobic deterioration; however, when lactic acid is their only energy source, the ph will increase. The corn used in the present study had a low WSC content and the lactic acid contents in the nal silages were also relatively low. Since the corn silages had no residual WSC and little lactic acid which could serve as substrates for aerobic yeasts, all corn silages remained stable during the aerobic stability test. The wheat silages had high contents of both residual WSC and lactic acid and therefore, tended to spoil more upon aerobic exposure, as indicated by more intensive CO 2 production, but without change in ph. In the current study, the aerobic stability test of wheat silage was conducted at both room temperature (26 C, in June) and at 33 C. Another study in this laboratory, in which aerobic stability tests of commercial wheat and corn silages were performed at 40 C, resulted in stable silages because of inactivation of aerobic yeasts and moulds at that temperature; the most intensive aerobic spoilage occurred at 20 and 30 C (Ashbell et al. submitted for publication). Furthermore, a Table 6 Aerobic stability (AS) test of the wheat silages. Yeasts and moulds are given as log cfu g )1 Treatment Silage temperature AS test temperature CO 2 gkg )1 DM ph Yeasts Moulds Control 24 C 26 C 0 3á9 0á0 2á4 <2á0 33 C 0 3á9 0á1 8á8 <2á0 41 C 26 C 0 4á1 0á0 7á3 <2á0 33 C 4á3 1á1 4á1 0á0 8á1 5á2 Inoculant 24 C 26 C 0 3á9 0á0 7á5 2á5 33 C 2á5 1á0 3á9 0á0 6á7 3á0 41 C 26 C 0 4á1 0á0 5á7 3á3 33 C 5á3 1á2 4á1 0á0 5á9 6á3 Both ensiling temperature and aerobic stability temperature had signi cant effects on CO 2 production (P < 0á05).

6 566 Z.G. WEINBERG ET AL. Table 7 Acid detergent bre (ADF) and acid detergent insoluble nitrogen (ADIN) of the corn and wheat control silages, g kg )1 (DM) Crop Temperature ADF ADIN Corn Fresh material C C ±37±28 C Wheat Fresh material C C temperature of 30±35 C is often measured at the face of normal silages in Israel. Therefore, 33 C was chosen as the high temperature in the aerobic stability test. The results reveal that aerobic deterioration of the wheat silages was more intensive when the silage had a high temperature history and the test, too, was performed at elevated temperatures. Therefore, special care should be taken during silage making and unloading in a warm climate in order to minimize temperature increase and consequent losses. The inoculated wheat silages tended to spoil more than the respective control silages, as often happens when wheat silages are inoculated with homofermentative LAB, due to lack of enough volatile fatty acids which inhibit fungi (Weinberg et al. 1993). Ammonia-N and ADIN contents were measured in an attempt to follow potential changes in nutritive value which might have resulted from elevated temperatures in the silages. Ammonia-N represents protein breakdown and ADIN represents insoluble nitrogen that might have been produced from any heat denaturation process (the temperatures used in the present study were too low and the moisture content too high for browning types of reactions). Except for somewhat higher ammonia-n content in the corn silages stored at the high temperatures (which agrees with Muck and Dickerson 1988), no differences between temperature treatments with regard to ADF and ADIN contents were observed. This is in agreement with the ndings of McDonald et al. (1966) who did not nd any differences in nutritive value among grass silages stored at different temperatures and fed to sheep. Conclusions High temperatures (37±41 C) during ensiling of whole crop corn and wheat result in higher ph, less lactic acid and greater losses than cooler temperatures (24±28 C). Warmer silages might also be more susceptible to aerobic deterioration, especially if the ambient temperature is high. Therefore, in a warm climate, special care should be taken during all stages of silage making. The ef cacy of inoculants for silage might also be affected by high ensiling temperatures, and this point should be considered during their formulation. ACKNOWLEDGEMENTS Contribution from the Agricultural Research Organization, The Volcani Center, Bet Dagan, Israel, no. 421-E, 2000 series, is acknowledged. REFERENCES Ashbell, G., Weinberg, Z.G., Azrieli, A., Hen, Y. and Horev, B. (1991) A simple system to study the aerobic deterioration of silages. 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