Printer Friendly

Dynamics of change in fermentation and fatty acid profiles in high moisture alfalfa silage during ensiling at different temperatures/ A dinamica de mudanca na fermentacao e perfis de acidos graxos em silagem de alfafa de alta humanidade durante a ensilagem a diferentes temperaturas.


Fatty acids (FA) in fresh forage is dominated by a high proportion of linoleic acid (C18:2n-6) and a-linolenic acid (C18:3n-3) (CLAPHAM et al., 2005). High intake ration containing sufficient fresh forage can increase the concentration of C18:2n-6 and C18:3n-3 in ruminant products and consequently be beneficial to human health (SIMOPOULOS, 2001). Fresh forage silage is increasingly fed to ruminant in many regions of the world. FA in silages, mainly dynamics of change in C18:2n-6 and C18:3n-3, has gained high attention during ensiling (ALVES et al., 2011).

Previous studies have reported the increase or reduction in FA of silage as compared with fresh forage (BOUFAIED et al., 2003; ELGERSMA et al., 2003; ARVIDSSON et al., 2009; VAN RANST et al., 2009; ALVES et al., 2011). However, the real reason is not completely known. One explanation is that plant enzymes and microbes play role in FA change during ensiling (ELGERSMA et al., 2003; DING et al., 2013). Plant lipases and lipoxygenases are the main enzymes responsible for change of FA because plant lipases release FA from damaged tissues after cutting and followed ensiling (CHOW et al., 2004) and plant lipoxygenases oxidize FA (FEUSSNER & WASTERNACK, 2002). Activity of enzymes and microbes is influenced by temperature (MCDONALD et al., 1991; LIU et al., 2016). However, until now few publications are related to plant enzymes and microbes in alfalfa silage ensiled at different temperatures, which may provide important information for further regulation of fermentation and FA.

The purpose of this study was to investigate dynamics of change in fermentation and FA profiles in high moisture alfalfa silage during ensiling at 15[degrees]C, 30[degrees]C and 45[degrees]C for 65 days, and the activity of lipase and lipoxygenase at different pH values and temperatures in a simulative ensiling system.


Silage material and silage making

Alfalfa (Medicago sativa cv. Jili) was planted on September 25, 2014, in a field of Nanjing Agricultural University (Nanjing, China). At the early flowering stage on April 15, 2015, alfalfa (DM=232 g/kg FW) was harvested for making silage. Alfalfa was chopped into 1 to 2 cm-long pieces by a forage chopper (Sh-2000, Shanghai Donxe Industrial Co., Ltd., Shanghai, China).

An experiment on alfalfa silage ensiled at 3 temperatures (15[degrees]C, 30[degrees]C and 45[degrees]C) x 7 ensiling times (0, 1, 3, 7, 21, 39 and 65 days) x 4 replicates was designed. Chopped fresh alfalfa was mixed well and subdivided into 72 smaller batches. The weight of each batch was 750g, with each batch corresponding to one plastic laboratory silo (1000mL capacity). The silo was filled with a batch and sealed with a screw top and plastic tape. Three incubators were used to achieve the ensiling temperatures of 15[degrees]C, 30[degrees]C and 45[degrees]C. Subsequently, randomly selected 24 silos were kept in the corresponding incubator. After ensiling for 1, 3, 7, 21, 39 and 65 days, randomly selected 4 silos in each incubator were opened.

Microbial and chemical analyses

The microorganism numbers in the fresh materials and silages were determined by the plate count method (DING et al., 2013). Ten grams of the fresh alfalfa was shaken well with 90mL of sterilized saline solution (8.50g/L NaCl) and serial dilutions ([10.sup.-1] through [10.sup.-7]) were made in sterile saline solution. Lactic acid bacteria (LAB) was counted on deMan Rogosa and Sharp agar medium (Difco Laboratories, Detroit, MI, USA) after incubation in an anaerobic incubator ([N.sub.2]: [H.sub.2]: C[O.sub.2] = 85:5:10, YQX-IL CIMO Medical Instrument Manufacturing Co., Ltd, Shanghai, China) at 37[degrees]C for 2 days. Aerobic bacteria were cultured and counted on nutrient agar medium (Guangdong Huankai Microbial Science and Technology Co., Ltd., Guangzhou, China), yeasts were counted on potato dextrose agar (Guangdong Huankai Microbial Science and Technology Co., Ltd., Guangzhou, China) acidified with a sterilized tartaric acid solution to pH 3.5. The agar plates were incubated at 37[degrees]C for 2 days. All microbial data were transformed to [log.sub.10] and presented on a wet weight basis.

Fifty grams of silage was taken after ensiling, mixed with 200mL of distilled water, and stored at 4[degrees]C for 18 hours. The mixture was then filtered, and the filtrate was used for measuring pH value using a glass electrode pH meter (HI221, Hanna Ltd., Rome, Italy). The buffering capacity, contents of dry matter (DM), water-soluble carbohydrates (WSC), crude protein (CP), neutral detergent fiber (NDF) and acid detergent fiber (ADF) of alfalfa material or silage was determined using the same methods in our previous study (LIU et al., 2016). After ensiling for 1, 3, 7, 21, 39 and 65 days, silos were opened and the silages were mixed thoroughly. Silages were sampled, and DM and microbes of silage were measured by the same method with the fresh alfalfa. The filtrate of silage was used for measuring pH value, contents of ammonia-N and organic acid. Contents of ammonia-N and organic acids (lactic acid, acetic acid, propionic acid and butyric acid) were determined by the same procedures in our previous study (LIU et al., 2016).

FA analysis

Lipids were extracted using a slightly modified version of the method described by Folch et al. (1957). Briefly, the 1 g frozen dried sample was added with 5mL preheated isopropanol in a glass tube, heated at 75[degrees]C for 15min, and then cooled to room temperature. Glass tube was added with 3mL chloroform and 1mL water and was incubated with shaking for 60 minutes. Liquid extract was transferred to a fresh tube, extract tissue was added with 4.5mL chloroform: methanol (2:1 v/v) two times until tissues are grayish white. All extracts were combined and added 2mL 1mol/L KCl. After mixing, the extracts were centrifuged at 1820g at 16[degrees]C, and 16mL lower phase was obtained. After concentrated by Termovap sample concentrator (MD200-2, Allsheng Instruments Co., Ltd., Hangzhou, China) at 45[degrees]C, the 2mL sample was added 2mg of nonadecanoic acid (C19:0; Sigma, Shanghai, China) as internal standard and 5mL of 2.5% H2SO4 (v/v) in methanol, and then heated at 80[degrees]C for 60 minutes to methyl esterification. A 1.5mL of heptane was added and followed by 1mL 0.9% NaCl (w/v) to extract fatty acids methyl esters (FAME).

FAME was analyzed on Agilent 7890A gas chromatograph (Agilent Technologies Inc., Munchen, Germany) with a capillary column HP-88 (100m x 0.25mm i.d. x 0.2 [micro]m, Agilent Technologies Inc., Shanghai, China). The temperature program was used: 150[degrees]C for 2min, followed by an increase at a rate of 0.8[degrees]C/min until 220[degrees]C. Temperatures of the injector and detector were 250[degrees]C, respectively. A FAME mixture obtained from Sigma (Supelco 37 component, Supelco Inc. Bellefonte, PA, USA) was used as a standard to quantify individual FA.

Activity of lipase and lipoxygenase analyses in simulative ensiling system

Reagents were prepared using lactic acid, acetic acid, butyric acid and ammonium oxalate for simulating ensiling system with diffent pHs according to the fermentation profiles of silage (Table 1). The activities of lipase and lipoxygenase in fresh leaves of alfalfa and at diffent reagents and temperatures were analyzed by Plant Lipase and Lipoxygenase Activity Kit (Shanghai Cablebridge Biotechnology Co., Ltd., Shanghai, China) according to the method in MALEKIAN et al. (2000).

Statistical analyses

The statistical analyses were performed using the IBM Statistical Packages for the Social Sciences (IBM SPSS 20.0 for Windows). Repeated measures analysis of variance (General Linear Models) (3 temperatures x 6 ensiling times x 4 replicates) were used to evaluate effects of temperatures, ensiling time and their interactions on the fermentation characteristics and microbial compositions in high moisture alfalfa silages. Repeated measures analysis of variance (General Linear Models) (3 temperatures x 7 ensiling times x 4 replicates) were used to evaluate effects of temperatures, ensiling time and their interactions on FA profile in high moisture alfalfa silages. The data were analyzed by two-way ANOVA (4 pHs x3 temperatures x3 replicates) to evaluate effects of pH, temperatures and their interactions on the activity of lipase and lipoxygenase, respectively. The data were analyzed by one-way ANOVA (3 temperatures x4 replicates) to evaluate of temperatures on chemical composition of alfalfa silage after ensiling for 65 days. The means were then compared for significance using Tukey's test at P < 0.05.


Characteristics of alfalfa before ensiling

The present study showed that alfalfa had low contents of DM (232g/kg FW) and WSC (50.8g/ kg DM), and had high crude protein content (180g/kg DM), NDF (404g/kg DM) and ADF (317g/kg DM) content and buffering capacity (226mEq/kg DM). Epiphytic LAB (5.92[log.sub.10] cfu/g FM) was less than aerobic bacteria (6.68[log.sub.10] cfu/g FM) and yeasts (6.71[log.sub.10] cfu/g FM). In addition, the activity of lipase (1.05U/100mg) in fresh alfalfa was lower than lipoxygenase (16.6U/100mg).

Effect of temperature on dynamic of change in fermentation characteristics

Lactic fermentation changing into butyric fermentation in silage stored at 30[degrees]C and 45[degrees]C occurred on ensiling for 21 and 65 days, respectively, as accompanied with high ammonia-N content (>150g/kg N) (Table 2), and resulted in a sudden increase in pH of silage (P < 0.05). This indicated protein degradation and poor fermentation quality. In contrast, the increase of lactic acid content was observed in silage ensiled at 15[degrees]C during ensiling for 65 days (P < 0.05). This can be attributed to the eco-physiological properties of variational microflora in alfalfa ensiling at different temperatures and stages, supported by the LAB number, pH, contents of lactic acid, butyric acid and ammonia-N were influenced by the interaction of temperature and ensiling time (P < 0.05) (Table 3). CAO et al. (2011) and WANG & NISHINO (2013) reported that prolonging storage time of silage tended to decrease pH and to increase lactic acid content and amount of LAB at low temperature. Most clostridia species are mesophilic bacteria and vigorous butyric fermentation is often found in the middle and later stages of ensiling (MCDONALD et al., 1991). LIU et al. (2011) reported that the vigorous butyric fermentation with high pH and ammonia-N content was at 30[degrees]C after ensiling for 45 days. Butyric fermentation is sometimes associated with high acetic acid production because of proteolytic clostridia producing ammonia-N, acetic acid and butyric acid from peptides and amino acids (PAHLOW et al., 2003). This was confirmed in the present study which showed sudden increases in contents of acetic acid and ammonia-N (P < 0.05) when silage ensiled at 30[degrees]C and 45[degrees]C separately occurred vigorous butyric fermentation. However, the acetic acid content of silage ensiled at 15[degrees]C, with weak butyric fermentation, still increased as prolonging the ensiling time (P < 0.05). Especially, acetic acid content was 2.6 times higher than lactic acid content after ensiling for 65 days and is accompanied with high ammonia-N (>120g/kg N). Based on the present study, compared with at 45[degrees]C and 30[degrees]C, higher LAB and aerobic bacteria number might be responsible for acetic fermentation and ammonia-N formation at 15[degrees]C (P < 0.05). Some researchers proposed that: facultatively heterofermentative LAB and enterobacteria were contributors for acetic fermentation and ammonia-N formation, e.g., Lactobacillusplantarum can deaminate serine to produce acetic acid and ammonia-N (LIU et al., 2003; PARVIN & NISHINO, 2009); Alfalfa and ryegrass silage with many Hafnia alvei was vigorous acetic fermentation and had high ammonia-N content (>100g/kg N) (MCDONALD et al., 1991).

Effect of temperature on chemical composition of final silage

Silage ensiled at 15[degrees]C had higher CP content (P < 0.05) and lower contents of NDF (P < 0.05 or 0.05 < P < 0.1) and ADF (P < 0.05) than at 30[degrees]C and 45[degrees]C, since fermentation qualities of silage at 30[degrees]C and 45[degrees]C were poorer than at 15[degrees]C (Figure 1). Similar with previous studies (MCDONALD et al., 1991; LIU et al., 2012), clostridia degraded most nutrients and leave cell wall residue in poor quality silage.

Effect of temperature on dynamic of change in FA profile

As shown in table 3, C16:0, C18:2n6 and C18:3n3 were the main FA composition in fresh alfalfa. Temperature influenced contents of total FA, C18:2n6 and C18:3n3 in silage during ensiling (P < 0.05). Compared with alfalfa before ensiling, there was a considerable loss of total FA content in silage after ensiling 65 days (P < 0.05). This was attributed to a fact that C18:2n-6 and C18:3n-3 lipolysis mainly caused the loss of FA despite there was an increase in C16:0 content during ensiling at any temperatures (P<0.05), which was similar to the results in previous studies (DING et al., 2013; KE et al., 2015). HAN & ZHOU (2013) found that the C18:2n-6 and C18:3n-3 losses in silages were mainly due to the activity of lipoxygenase. Further results showed that activity of lipoxygenase responsible for FA lipolysis was higher than lipase responsible for FA formation at each pH and temperature in the simulative ensiling system (P < 0.05) (Figure 2).

As the ensiling temperature rose, losses in contents of total FA, C18:2n6 and C18:3n3 increased after ensiling for 1 day as compared with alfalfa before ensiling (P < 0.05) (Table 3). This could be attributed to the fact that multiplication of epiphytic LAB was promoted at a high ensiling temperature at the initial stage of ensiling, supported by higher LAB number (8.37[log.sub.10] cfu/g FM) at 45[degrees]C than at 30[degrees]C and 15[degrees]C on ensiling for 1 day (P < 0.05). As the temperature rose in the simulative ensiling system, the activity of lipoxygenase decreased (P < 0.05) (Figure 2), but the total FA, C18:2n-6 and C18:3n-3 lipolysis in silage was not restrained after ensiling for one day. This indicated that intervention of lipoxygenase was few in FA lipolysis at 45[degrees]C. Actually, epiphytic LAB have the ability to bio-hydrogenate C18:2n-6 and C18:3n-3 (OGAWA et al., 2005; KISHINO et al., 2009), and their activity of biohydrogenation could be enhanced by increasing temperature (TAKEUCHI et al., 2015). KUMARATHASAN et al. (1992) and JUITA et al. (2012) reported that polyunsaturated acids, such as C18:2n-6 and C18:3n-3, undergo rapid oxidation at elevated temperature.

During ensiling from 3 to 65 days, contents of total FA, C18:2n-6 and C18:3n-3 fluctuated in silage ensiled at 30[degrees]C and 45[degrees]C, but decreased in silage ensiled at 15[degrees]C (P < 0.05) (Table 3). Therefore, after ensiling for 65 days, silages maintained at 15[degrees]C had lower contents of total FA and C18:2n-6 than silages maintained at 30[degrees]C (P < 0.05) and 45[degrees]C (0.05 < P < 0.1), and had lower content of C18:3n-3 than silages maintained at 30[degrees]C and 45[degrees]C (P < 0.05). The former did not support the hypothesis of VAN RANST et al. (2009), who reported that total FA content remained stable irrespective of type and extent of fermentation. Regrettably, its reason was not yet elaborated. Fluctuation of contents of total FA, C18:2n-6 and C18:3n-3 in silage ensiled at 30[degrees]C and 45[degrees]C during ensiling from 3 to 65 days possibly was attributed to the intervention of different microbes and plant enzymes but which need further study. To our best knowledge, there were few reports for the decrease in contents of total FA, C18:2n-6 and C18:3n-3 in silage ensiled at 15[degrees]C. Based on our results, this could be attributed to the activity of microbes, showed by higher amount of aerobic bacteria and yeasts on most of ensiling days at 15[degrees]C than at 30[degrees]C and 45[degrees]C (P < 0.05 or 0.05 < P < 0.1), and the higher activity of lipoxygenase at 15[degrees]C than at 30[degrees]C and 45[degrees]C (P < 0.05) (Figure 2). A significant interaction of temperature and ensiling time was on C16:0 (P < 0.05), shown by higher C16:0 content at 15[degrees]C and 30[degrees]C than 45[degrees]C during ensiling for 65 days (P < 0.05). This might be attributed to that C16:0 responded to different micro-ecologies during ensiling, supported by lower aerobic bacteria and yeasts number at 45[degrees]C than at 15[degrees]C and 30[degrees]C (P < 0.05). This was similar to the result of ALVES et al. (2011), who reported that the use of formic acid as fermentation-inhibitor decreased microbial FA synthesis, and thus which made a decrease in the C16:0 as compared with the control.

In conclusion, after ensiling for 65 days, alfalfa silage ensiling at low temperature (15[degrees]C) had better fermentation quality than at high temperatures (30[degrees]C and 45[degrees]C). Temperatures could induce various interventions of plant enzymes and microbes in FA lipolysis, which resulted in different dynamics of change in FA profile in alfalfa silage during ensiling.

Received 08.29.17 Approved 12.26.17 Returned by the author 02.25.18


The Project was supported by National Natural Science Foundation of China (31502014), Supported by the Fundamental Research Funds for the Central Universities (KJQ[N.sub.2]01603 and Y0201500224), The National Key Research and Development Program of China (2017YFD0502106-1), Key research project of Tibet (2017ZDKJZC-14).


ALVES, S.P., et al. Effect of ensiling and silage additives on fatty acid composition of ryegrass and corn experimental silages. Journal of Animal Science, v.89, n.8, p.2537-45. 2011. Available from: <>. Accessed: Mar. 11, 2011. doi: 10.2527/jas.2010-3128.

ARVIDSSON, K., et al. Effects of conservation method on fatty acid composition of silage. Animal Feed Science and Technology, v.148, n.2-4, p.241-252. 2009. Available from: <https://doi. org/10.1016/j.anifeedsci.2008.04.003>. Accessed: Jan. 16, 2009. doi: 10.1016/j.anifeedsci.2008.04.003.

BOUFAIED, H., et al. Fatty acids in forages. I. Factors affecting concentrations. Canadian Journal of Animal Science, v.83, n.3, p.501-511. 2003. Available from: <>. Accessed: Sep. 01, 2003. doi: 10.4141/A02-098.

CAO, Y., et al. Fermentation characteristics and microorganism composition of total mixed ration silage with local food byproducts in different seasons. Animal Science Journal, v.82, n.2, p.259-266. 2011. Available from: < 11/j.1740-0929.2010.00840.x>. Accessed: Mar. 2, 2011. doi: 10.1111/j.1740-0929.2010.00840.x.

CHOW, T.T., et al. Fatty acid content, composition and lipolysis during wilting and ensiling of perennial ryegrass (Lolium perenne L.): preliminary findings. Land use systems in grassland dominated-regions. In: Proceedings of the 20th General Meeting of the European Grassland Federation, Switzerland: Luzern, 2004, p. 21-24.

CLAPHAM, W.M., et al. Fatty acid composition of traditional and novel forages. Journal of Agricultural and Food Chemistry, v.53, n.26, p. 10068-10073. 2005. Available from: <https://doi. org/10.1021/jf0517039>. Dec. 01, 2005. doi: 10.1021/jf0517039.

DING, W.R., et al. Effects of plant enzyme inactivation or sterilization on lipolysis and proteolysis in alfalfa silage. Journal of Dairy Science, v.96, n.4, p.2536-2543. 2013. Available from: <>. Accessed: Feb. 15, 2013. doi: 10.3168/jds.2012-6438.

ELGERSMA, A., et al. Comparison of the fatty acid composition of fresh and ensiled perennial ryegrass (Lolium perenne L.), affected by cultivar and regrowth interval. Animal Feed Science and Technology, v.108, n.1-4, p.191205. 2003. Available from: < (03)00134-2>. Accessed: Aug. 01, 2003. doi: 10.1016/ S0377-8401(03)00134-2.

FEUSSNER, I.; WASTERNACK, C. The lipoxygenase pathway. Annual Review of Plant Biology, v.53, p.275297. 2002. Available from: < arplant.53.100301.135248>. Accessed: Jan. 01, 2002. doi: 10.1146/ annurev.arplant.53.100301.135248.

FOLCH, J., LEES, M., SLOANE-STANLEY G.H. A simple method for the isolation and purification of total lipids from animal tissues. Journal of Biological Chemistry, v. 226, p.497-509. 1957. Available from: < content/226/1/497.long>. Accessed: Aug. 23, 1956. doi: http://

HAN, L.Y.; ZHOU, H. Effects of ensiling processes and antioxidants on fatty acid concentrations and compositions in corn silages. Journal of Animal Science and Biotechnology, v.4. 2013. Available from: <>. Accessed: Dec. 04, 2013. doi: Artn 4810.1186/2049-1891-4-48.

KE, W.C., et al. Fermentation characteristics, aerobic stability, proteolysis and lipid composition of alfalfa silage ensiled with apple or grape pomace. Animal Feed Science and Technology, v.202, p.12-19. 2015. Available from: < anifeedsci.2015.01.009>. Accessed: Jan. 17, 2015. doi: 10.1016/j. anifeedsci.2015.01.009.

LIU, Q.H., et al. Characteristics of isolated lactic acid bacteria and their effectiveness to improve stylo (Stylosanthes guianensis Sw.) silage quality at various temperatures. Animal Science Journal, v.83, n.2, p. 128-135. 2012. Available from: <https://www.ncbi.>. Accessed: Aug. 16, 2011. doi: 10.1111/j.1740-0929.2011.00937.x.

LIU, Q.H., et al. The effect of fibrolytic enzyme, Lactobacillus plantarum and two food antioxidants on the fermentation quality, alpha-tocopherol and beta-carotene of high moisture napier grass silage ensiled at different temperatures. Animal Feed Science and Technology, v.221, p.1-11. 2016. Available from: <https://doi. org/10.1016/j.anifeedsci.2016.08.020>. Accessed: Aug. 24, 2016. doi: 10.1016/j.anifeedsci.2016.08.020.

LIU, Q. H., et al. The effects of wilting and storage temperatures on the fermentation quality and aerobic stability of stylo silage. Animal Science Journal, v.82, n.4, p.549-553. 2011. Available from: < 111/j.1740-0929.2011.00873.x>. Accessed: Apr. 20, 2011. doi: 10.1111/j.1740-0929.2011.00873.x.

LIU, S.Q., et al. Serine metabolism in Lactobacillus plantarum. International Journal of Food Microbiology, v.89, n.2-3, p.265-273. 2003. Available from: <>. Accessed: Dec. 01, 2003. doi: 10.1016/S01681605(03)00157-0.

MCDONALD, P., et al. Microorganism. In: The Biochemistry of Silage. 2ed. Aberystwyth: Cambran printers Ltd, 1991. Chap. 4. p.81-151.

MALEKIAN, F. et al. Lipase and lipoxygenase activity, functionality, and nutrient losses in rice bran during storage. Bulletin of the Louisiana Agricultural Experiment Station, v. 870, 69 p. 2000.

PAHLOW, G. et al. Microbiology of ensiling. In: Silage science and technology. BUXTON, D.R; MUCK, R.E.; HARRISON, J.H. American Society of Agronomy, USA: Madison, Wisconsin, 2003, pp.31-93.

PARVIN, S.; NISHINO, N. Bacterial community associated with ensilage process of wilted guinea grass. Journal of Applied Microbiology, v. 107, n.6, p.2029-2036. 2009. Available from: <>. Accessed: May 20, 2009. doi: 10.1111/j.1365-2672.2009.04391.x.

SIMOPOULOS, A.P. n-3 fatty acids and human health: Defining strategies for public policy. Lipids, v.36, p.S83-S89. 2001. Available from: <>. Accessed: Jan. 01, 2001. doi: 10.1007/s11745-001-0687-7.

VAN RANST, G., et al. Influence of ensiling forages at different dry matters and silage additives on lipid metabolism and fatty acid composition. Animal Feed Science and Technology, v.150, n.1-2, p.62-74. 2009. Available from: < anifeedsci.2008.08.004>. Accessed: Mar. 01, 2009. doi: 10.1016/j. anifeedsci.2008.08.004.

WANG, C.; NISHINO, N. Effects of storage temperature and ensiling period on fermentation products, aerobic stability and microbial communities of total mixed ration silage. Journal of Applied Microbiology, v.114, n.6, p.1687-1695. 2013. Available from: <>. Accessed: Apr. 09, 2013. doi: 10.1111/jam.12200.

Qinhua Liu (1) Zhihao Dong (1) Tao Shao (1) *

(1) Institute of Ensiling and Processing of Grass, Nanjing Agricultural University, Weigang 1, 210095, Nanjing, China. E-mail: * Corresponding author.

Caption: Figure 1--Effect of temperatures on chemical composition of alfalfa silage after ensiling for 65 days. Means with different lowercase letters above the column (a-d) indicated a significant difference according to Tukey's test at P < 0.05.

Caption: Figure 2--The activity of lipoxygenase and lipase at different pH values and temperatures. Means with different lowercase letters above the column (a-d) indicated a significant difference according to Tukey's test at P <0.05.
Table 1--Reagents with different pH values.

Ingredients                                                   pH

                                                          4.5     5.0
Lactic acid aqueous solution 85:100 (w/v, [micro]L)       15       5
Acetic acid aqueous solution 99.5:100 (w/v, [micro]L)     35      25
Butyric acid aqueous solution 98:100 (w/v, [micro]L)      20      50
Ammonium oxalate aqueous solution 6.21:100 (w/v, mL)      5.5      8
Distilled water (mL)                                     14.43   11.92

Ingredients                                                   pH

                                                          5.5     6.0
Lactic acid aqueous solution 85:100 (w/v, [micro]L)       2.5     2.5
Acetic acid aqueous solution 99.5:100 (w/v, [micro]L)     10      2.5
Butyric acid aqueous solution 98:100 (w/v, [micro]L)      100      0
Ammonium oxalate aqueous solution 6.21:100 (w/v, mL)       9       1
Distilled water (mL)                                     10.90   19.00

Table 2--Effects of temperature on dynamics of change in fermentation
characteristics and microbal composition of alfalfa silage during
ensiling for 65 days.

Items (a)              Tem                                  ET (d)

                                    1       3        7        21

pH                 45[degrees]C   5.92     4.94     4.95     4.88
                   30[degrees]C   6.05     5.57     5.29     5.70
                   15[degrees]C   6.28     6.24     5.87     5.75

Means of ET                       6.08     5.58     5.37     5.44

Lactic acid        45[degrees]C   3.43    22. 7     16.5     23.8
(g/kg DM)          30[degrees]C   13.1     22.3     25.0     0.76
                   15[degrees]C   0.56     9.87     13.7     10.5

Means of ET                       5.70     16.1     18.4     11.7

Acetic acid        45[degrees]C   1.06     3.50     3.66     3.61
(g/kg DM)          30[degrees]C   11.2     17.8     24.7     32.1
                   15[degrees]C   2.87     10.1     13.7     17.8

Means of ET                       5.04D   10.5CD   14.0BC   17.8BC

Propionic          45[degrees]C   0.13     0.40     0.27     0.30
acid (g/kg         30[degrees]C   0.43     0.23     1.50     6.50
DM)                15[degrees]C   0.13     0.27     0.33     0.07

Means of ET                       0.23     0.30     0.70     2.29

Butyric            45[degrees]C   0.30     1.80     0.93     3.90
acid (g/kg         30[degrees]C   0.83     1.03     1.37     30.9
DM)                15[degrees]C   0.07     0.20     0.80     0.37

Means of ET                       0.40     1.01     1.03     11.7

Ammonia-           45[degrees]C   63.4     86.9     72.3     79.8
N (g/kg N)         30[degrees]C   88.2     154      145      197
                   15[degrees]C   46.4     109      98.7     133

Means of ET                       66.0     117      105      137

LAB ([log.sub.10   45[degrees]C   8.37     6.66     7.03     5.22
cfu/FM)            30[degrees]C   7.94     7.70     7.96     7.51
                   15[degrees]C   7.54     7.44     7.58     8.25

Means of ET                       7.95     7.27     7.52     6.99

Aerobic            45[degrees]C   6.20     5.10     4.30     3.32
bacteria           30[degrees]C   7.68     6.52     5.58     5.22
([log.sub.10]      15[degrees]C   8.50     7.20     6.35     5.39

Means of ET                       7.46A   6.27B    5.41C    4.64D

Yeasts             45[degrees]C   <2.00   <2.00    <2.00    <2.00
([log.sub.10]      30[degrees]C   4.38     2.00     2.91    <2.00
cfu/FM)            15[degrees]C   3.78     3.62     2.79     4.92

Means of ET                       3.39     2.54     2.57     2.97

Items (a)                           Means             Significance
                                    of Tem    SEM
                     39      65                       Tem       ET

pH                  4.83    5.81     5.22    0.056   0.001    <0.001
                    5.73    5.45     5.63
                    5.56    5.39     5.85

Means of ET         5.37    5.55

Lactic acid         25.6    8.35     15.5    0.603   <0.001   <0.001
(g/kg DM)           0.52    2.20     10.7
                    12.7    14.2     10.3

Means of ET         12.9    8.26

Acetic acid         5.71    20.5    6.35 c   1.371   <0.001   <0.001
(g/kg DM)           25.6    53.9    27.6 a
                    28.9    36.3    18.3 b

Means of ET        20.1B    36.9A

Propionic           0.40    4.33     0.97    0.611   0.002    <0.001
acid (g/kg          10.6    15.1     5.73
DM)                 0.40    1.70     0.48

Means of ET         3.80    7.04

Butyric             4.83    20.4     5.36    1.165   <0.001   <0.001
acid (g/kg          36.4    38.9     18.2
DM)                 0.01    1.30     0.46

Means of ET         13.8    20.2

Ammonia-            106      240     108     8.056   0.006    <0.001
N (g/kg N)          192      197     162
                    163      128     113

Means of ET         154      188

LAB ([log.sub.10    4.89    5.48     6.28    0.050   <0.001   0.001
cfu/FM)             7.60    6.95     7.61
                    7.72    8.25     7.80

Means of ET         6.74    6.89

Aerobic             3.11    2.11    4.02c    0.076   <0.001   <0.001
bacteria            4.11    4.15    5.54b
([log.sub.10]       5.48    5.61    6.42a

Means of ET        4.23DE   3.96E

Yeasts             <2.00    <2.00    2.00    0.086   <0.001   0.008
([log.sub.10]      <2.00    <2.00    2.55
cfu/FM)             2.45    3.55     3.52

Means of ET         2.15    2.52

Items (a)          Significance

                   Tem x ET

pH                  <0.001

Means of ET

Lactic acid         <0.001
(g/kg DM)

Means of ET

Acetic acid         0.100
(g/kg DM)

Means of ET

Propionic           0.010
acid (g/kg

Means of ET

Butyric             <0.001
acid (g/kg

Means of ET

Ammonia-            <0.001
N (g/kg N)

Means of ET

LAB ([log.sub.10    <0.001

Means of ET

Aerobic             0.071

Means of ET

Yeasts              0.002

Means of ET

Means with different lowercase letters in the same column (a-c) or
capital letters (A-E) in the same row indicated a significant
difference according to Tukey's test at P < 0.05. (a) cfu, colony-
forming units; DM, dry matter; ET, ensiling time; FM, fresh matter;
LAB, lactic acid bacteria; N, nitrogen; SEM, standard error of the
means; Tem, temperature; Tem x ET, the interaction of temperature and
ensiling time.

Table 3--Effects of temperature on dynamics of change in the contents
(g/kg DM) of total fatty acid and detected fatty acids during ensiling
for 65 days.

Items (a)         Tem                        ET (d)

                               0       1       3       7      21

Total         45[degrees]C   33.8    20.1    23.9    20.7    22.8
FA            30[degrees]C   33.8    25.0    26.3    27.7    25.7
              15[degrees]C   33.8    31.1    29.3    26.8    27.4

Means of ET                  33.8A   25.4B   26.5B   25.1B   25.3B

C16:0         45[degrees]C   7.35    6.25    6.00    5.12    5.63
              30[degrees]C   7.35    9.30    9.73    9.30    11.10
              15[degrees]C   7.35    8.33    9.20    9.60    8.87

Means of ET                  7.35    7.96    8.31    8.01    8.53

C16:1         45[degrees]C   0.70    0.22    0.42    0.34    0.32
              30[degrees]C   0.70    0.63    0.63    0.70    0.50
              15[degrees]C   0.70    0.63    0.67    0.37    0.80

Means of ET                  0.70    0.49    0.57    0.47    0.54

C18:0         45[degrees]C   1.66    1.39    1.31    1.17    1.22
              30[degrees]C   1.66    1.47    1.50    1.70    1.47
              15[degrees]C   1.66    1.60    1.67    1.57    1.87

Means of ET                  1.66    1.48    1.49    1.48    1.52

C18:1         45[degrees]C   2.16    0.77    0.88    0.69    0.82
              30[degrees]C   2.16    2.00    1.43    1.47    1.43
              15[degrees]C   2.16    1.00    1.17    1.20    0.87

Means of ET                  2.16A   1.26B   1.16B   1.12B   1.04B

C18:2n6       45[degrees]C   5.99    4.87    5.66    5.11    5.50
              30[degrees]C   5.99    5.43    6.27    6.30    5.60
              15[degrees]C   5.99    7.80    6.50    7.17    6.10

Means of ET                  5.99    6.04    6.14    6.19    5.73

C18:3n3       45[degrees]C   15.9    6.57    9.65    8.22    9.36
              30[degrees]C   15.9    8.07    7.60    8.20    8.27
              15[degrees]C   15.9    9.77    9.30    6.90    6.10

Means of ET                  15.9A   8.13B   8.85B   7.77B   7.91B

Items (a)          ET (d)       Means              Significance
                               of Tem     SEM
               39       65                        Tem       ET

Total         23.9     23.1    24.0 b    0.616   0.007    <0.001
FA            30.7     26.2    27.9 a
              25.4     20.3    27.7 a

Means of ET   26.7B   23.2C

C16:0         6.03     8.17     6.36     0.199   <0.001   0.028
              10.13    8.77     9.38
              10.67    8.20     8.89

Means of ET   8.94     8.38

C16:1         0.37     0.57    0.42 b    0.042   0.024    0.305
              0.73     0.53    0.63 a
              0.53     0.53    0.60 a

Means of ET   0.55     0.54

C18:0         1.34     1.47     1.37     0.032   0.002    0.081
              1.80     1.37     1.57
              1.77     1.40     1.65

Means of ET   1.64     1.41

C18:1         0.80     1.03    1.02 b    0.084   0.014    0.001
              1.30     1.00    1.54 a
              1.43     0.90    1.25 ab

Means of ET   1.18B   0.98BC

C18:2n6       5.65     4.67    5.35 b    0.125   0.007    0.057
              6.40     5.40    5.91 a
              5.90     4.13    6.22 a

Means of ET   5.98     4.73

C18:3n3       9.68     7.13     9.50a    0.334   0.049    <0.001
              9.67     9.80     9.64a
              5.70     4.13     8.26b

Means of ET   8.35B   7.02C

Items (a)     Significance

              Tem x ET

Total          0.090

Means of ET

C16:0          <0.001

Means of ET

C16:1          0.403

Means of ET

C18:0          0.044

Means of ET

C18:1          0.723

Means of ET

C18:2n6        0.186

Means of ET

C18:3n3        0.056

Means of ET

Means with different lowercase letters in the same column (a-b) or
capital letters (A-C) in the same row indicated a significant
difference according to Tukey's test at P < 0.05. Total fatty acids
content was summed by each detected fatty acid. (a) ET, ensiling time;
FA, fatty acid; SEM, standard error of the means. Tem, temperature;
Tem x ET, the interaction of temperature and ensiling time.
COPYRIGHT 2018 Universidade Federal de Santa Maria
No portion of this article can be reproduced without the express written permission from the copyright holder.
Copyright 2018 Gale, Cengage Learning. All rights reserved.

Article Details
Printer friendly Cite/link Email Feedback
Author:Liu, Qinhua; Dong, Zhihao; Shao, Tao
Publication:Ciencia Rural
Date:Mar 1, 2018
Previous Article:Development and characterization of light yoghurt elaborated with Bifidobacterium animalis subsp. Lactis Bb-12 and...
Next Article:Enzymatic hydrolysis (pepsin) assisted by ultrasound in the functional properties of hydrolyzates from different collagens/ Hidrolise enzimatica...

Terms of use | Privacy policy | Copyright © 2020 Farlex, Inc. | Feedback | For webmasters