Animal Feed Science and Technology 288 (2022) 115301
Contents lists available at ScienceDirect
Animal Feed Science and Technology
journal homepage: www.elsevier.com/locate/anifeedsci
Antioxidant, flavonoid, α-tocopherol, β-carotene, fatty acids, and
fermentation profiles of alfalfa silage inoculated with novel
Lactiplantibacillus Plantarum and Pediococcus acidilactici strains
with high-antioxidant activity
X. Zhang a, c, X.S. Guo b, c, F.H. Li a, c, S. Usman a, Y.X. Zhang b, c, Z.T. Ding b, c, *
a
State Key Laboratory of Grassland Agro-ecosystems, College of Pastoral Agriculture Science and Technology, Lanzhou University, Lanzhou 730000,
PR China
b
School of Life Sciences, Lanzhou University, Lanzhou 730000, PR China
c
Probiotics and Bio-feed Research Center, Lanzhou University, Lanzhou 730000, PR China
A R T I C L E I N F O
A B S T R A C T
Keywords:
Antioxidant activity
Alfalfa silage
Fatty acids
Fermentation profiles
Lactic acid bacteria
Six lactic acid bacteria strains with high-antioxidant activity were screened and applied to alfalfa
silage to develop antioxidant probiotics for silage production. Alfalfa was ensiled without in­
oculants (control) or with a commercial strain (Lactiplantibacillus plantarum GFG) and antioxidant
strains L. plantarum 24-7 (24-7), L. plantarum BX62 (BX62), L. plantarum AS21 (AS21),
L. plantarum FM15 (FM15), Pediococcus acidilactici 13-7 (13-7), P. acidilactici J17 (J17) at dry
matter concentration of 400 g/kg for 60 d, respectively. Alfalfa silage was investigated on silage
fermentation, chemical and microbial compositions, antioxidant enzymes, total flavonoid,
α-tocopherol, β-carotene and fatty acid. The results showed that AS21, FM15, 13-7, and J17 in­
oculations improved (P < 0.001) lactic acid concentration in alfalfa silage compared with control
and GFG-treated silages. Compared with control and GFG-treated silages, AS21- (P < 0.001),
FM15- (P = 0.007), 13-7- (P = 0.03), and J17-treated (P < 0.001) silages decreased ammonia
nitrogen concentration. All inoculants with high-antioxidant activity reduced the losses of
α-tocopherol (P < 0.001) and β-carotene (P < 0.001). Higher total flavonoid concentration was
found in 24-7- (P = 0.004), BX62- (P < 0.001), FM15- (P = 0.03), J17-treated (P < 0.001) silages
versus control. In comparison to control and GFG-treated silages, 24-7- (P < 0.001), BX62- (P <
0.001), AS21- (P = 0.004), FM15- (P = 0.002), J17-treated (P < 0.001) silages had higher pro­
portion of polyunsaturated fatty acid. Except for strain J17, inoculations with the screened strains
had higher (P < 0.001) total antioxidant capacity (T-AOC) activity in alfalfa silage compared with
control and GFG-treated silages. The highest T-AOC (P < 0.001, 200 U/g FW) was observed in
Abbreviations: ADF, acid detergent fiber; CAT, catalase; CFU, colony forming unit; CP, crude protein; DM, dry matter; DPPH, 2, 2-diphenyl-1picrylhydrazyl; GSH-PX, glutathione peroxidase; H2O2, hydrogen peroxide; LAB, lactic acid bacteria; LOX, lipoxygenase; MRS, Man, Rogosa and
Sharpe; MUFA, monounsaturated fatty acid; aNDF, neutral detergent fiber assayed with a heat stable amylase and expressed inclusive of residual
ash; NPN, non-protein nitrogen; NH3-N, ammonia nitrogen; O−2 , superoxide anion; OH⋅, hydroxyl radicals; PUFA, polyunsaturated fatty acid; ROS,
reactive oxygen species; SFA, saturated fatty acid; SOD, superoxide dismutase; T-AOC, total antioxidant capacity; TCA, trichloroacetic acid; TFA,
total fatty acid; C16:0, palmitic acid; C18:1, oleic acid; C18:2, linoleic acid; C18:3, linolenic acid; c9,12,15C18:3, α-linolenic acid; WSC, water
soluble carbohydrates.
* Correspondence to: School of Life Sciences, Lanzhou University, No. 222 South Tianshui Road, Lanzhou 730000, PR China.
E-mail address: dingwr@lzu.edu.cn (Z.T. Ding).
https://doi.org/10.1016/j.anifeedsci.2022.115301
Received 7 November 2021; Received in revised form 15 April 2022; Accepted 16 April 2022
Available online 21 April 2022
0377-8401/© 2022 Elsevier B.V. All rights reserved.
Animal Feed Science and Technology 288 (2022) 115301
X. Zhang et al.
AS21-treated silage. Inoculated silages had higher (P < 0.001) glutathione peroxidase activity
versus control. Conversely, compared with control and GFG-treated silages, lower superoxide
dismutase activity was observed in FM15- (P < 0.001) and J17-treated (P < 0.001) silages. Lower
lipoxygenase (LOX) activity (P < 0.001) was observed in screened strains treated-silages
compared with control and GFG-treated silages. Pearson correlation analysis showed that TAOC was positively correlated with α-tocopherol (r = 0.66, P = 0.05) and β-carotene (r = 0.76, P
= 0.02), but negatively correlated with LOX (r = − 0.67, P = 0.05). Conclusively, all the tested
strains with high-antioxidant activity can be used as candidate strains to improve the antioxidant
status of ensiled alfalfa, and strain AS21 is the most preferred strain.
1. Introduction
Reactive oxygen species (ROS) usually evoke oxidative stress on animals which puts the animals in a sub-healthy state, thereby
affecting the productivity of the animal and ultimately causing production loss (Iqbal et al., 2012; Nisar et al., 2013). Previous studies
have shown that the application of exogenous antioxidants such as phenolic compounds, anthocyanins, catechin, pycnogenol,
astaxanthin, and flavonoids in the ruminant diet is an effective strategy to mitigate oxidative stress (Nisar et al., 2013; Pandey and
Negi, 2016; Khosravi et al., 2018). However, the application of these exogenous antioxidants as a feed additive could be limited by
their low output during extraction, lack of raw materials, and high extraction cost (Villena et al., 2011). In addition, with the
development of herbivorous animal husbandry, including large-scale dairy farms with over 133 million cattle worldwide (Jin et al.,
2015), approximately 667 million tons of silage are expected to be consumed per annum. Thus, there is an increasing interest in
producing silage with antioxidant properties for ruminant livestock (Zhang et al., 2020).
Lactic acid bacteria (LAB) and their metabolites have been confirmed by existing literature to have good antioxidant properties
(Kenfack et al., 2018). Meanwhile, the use of LAB as a feed additive has been widely adopted in animal production for efficient feed
utilization, diseases prevention, and enhancing the animals’ immune system (Yang et al., 2017). For instance, Ding et al. (2017) re­
ported that Lactobacillus delbrueckii subsp. bulgaricus F17 isolated from yak yogurt could effectively scavenge free radicals (59% hy­
droxyl radicals (OH⋅) and 54% superoxide anion (O-2) scavenged) by producing superoxide dismutase (SOD). LeBlanc et al. (2011) also
discovered that engineered L. casei BL23 strain produced either catalase (CAT) or SOD which increased enzymatic activities in the mice
gut, and relieved the intestinal inflammation. Based on these reports, it is established that most studies on the LAB antioxidant
properties focussed on the LAB itself and its application in food and animals. Thus, it is important to study the effect of LAB inoculant
on the antioxidant properties of silage and its subsequent effect after feeding the silage to animals.
The LAB has been widely applied in silage production to ensure a good fermentation quality (Sun et al., 2009). However, limited
studies are conducted to investigate the effects of antioxidant LAB strains on silage fermentation and antioxidant property. Only two of
our previous studies proved that application of antioxidant strain L. plantarum 24-7 or Pediococcus acidilactici J17 could improve the
antioxidant status of alfalfa silages at different dry matter (DM) contents, and significant effect was observed in alfalfa silage with DM
content of 400 g/kg versus 300 g/kg (Zhang et al., 2020, 2021). Hence, we hypothesized that application of LAB screened for
high-antioxidant activity on silage could promote fermentation quality and antioxidant status of the silage. Therefore, in the present
study, we evaluated the influences of six LAB strains (L. plantarum and P. acidilactici) with high-antioxidant properties, that were
previously screened in our laboratory, on the antioxidant status, chemical and fermentation properties of alfalfa ensiled for 60 d. The
aims were (1) to screen more LAB for probiotic production that could be applied to alfalfa silage, thereby developing silage with
antioxidant properties for ruminant livestock, (2) and to identify the most preferred strain for promoting silage fermentation quality
and antioxidant status in the six tested strains with high-antioxidant activity.
2. Materials and methods
2.1. Antioxidant properties of seven LAB strains
Six of the LAB strains used in this study were isolated from Elymus nutans silage produced in Nagqu, Qinghai-Tibetan plateau
(32◦ 57′ N, 117◦ 12′ E), out of which four strains are L. plantarum 24-7, BX62, FM15, AS21, and two strains P. acidilactici J17, 13-7. These
LAB strains with high-antioxidant activity were selected according to the method of Shimada et al. (1992) from 344 strains screened
using a high H2O2 (2 mmol/L) containing Man, Rogosa and Sharpe (MRS) medium. The DPPH, OH⋅, and O−2 of the screened strains
were determined according to the method of Ding et al. (2017). The T-AOC and SOD activities were measured using an assay kits
(Nanjing Jiancheng Bioengineering Research Institute Co., Ltd., Nanjing, China) according to the instruction manual. The commercial
strain L. plantarum GFG (CICC23108, China Center of Industrial Culture Collection, Beijing, China) was used as LAB control.
2.2. Silage preparation
Alfalfa (Medicago sativa L.) planted for four years was harvested by hand at early blooming stage as first-cut of the season from four
different fields (four plots per field), and wilted to DM concentration of about 400 g/kg on wet weight basis by natural drying. The
wilted alfalfa was then chopped into 1–2 cm lengths by a hay cutter (ZR-3, Panyue Machinery Equipment Co., Ltd., Henan, China)
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Animal Feed Science and Technology 288 (2022) 115301
X. Zhang et al.
immediately. The chopped forages from each field were divided into eight small piles after mixing thoroughly. A total of 32 forage piles
were prepared from the four fields (about 16 kg wilted forage), and the four sampling fields serve as replications for each treatment.
Completely randomized design was used to assign the prepared forage piles from each field to one of the following treatments: (1)
commercial strain L. plantarum GFG (GFG), (2) L. plantarum 24-7 (24-7), (3) L. plantarum BX62 (BX62), (4) L. plantarum AS21 (AS21),
(5) L. plantarum FM15 (FM15), (6) P. acidilactici 13-7 (13-7), (7) P. acidilactici J17 (J17), and (8) distilled water (control) making seven
treatments and control × four replicates. Additional fresh forage from the four fields (about 160 g wilted forage from each sampling
field, respectively) was collected as fresh alfalfa and frozen at − 20 ◦ C for further analysis. The application rate of each strain was 1 ×
105 colony forming units (CFU)/g fresh weight. Before application, all strains were enumerated by plate count method (up to 1010).
The strains were incubated in MRS medium for 8 h (logarithmic growth phase of the colony), and then the OD values (wavelength 600
nm) were measured to calculate the number of viable bacteria. Three replications of associating the OD results to cell counts for each
strain were done, and the replication process prior to the final culture was standardized. Before the silage preparation, these strains
were centrifuged for 5 min at 8000 × g and dissolved in a sterile distilled water to a concentration of 1 × 107 CFU/mL, respectively
(each strain was applied at the rate of 10 mL/kg fresh weight). The control had the same amount of sterile water applied to the forage
material. Finally, the prepared bacterium suspension and distilled water were evenly sprayed onto each pile using a micro atomizer
and thoroughly mixed in different plastic containers that had been previously disinfected with ethanol. All prepared samples were
packed in polyethylene plastic bags (dimensions: 270 mm × 400 mm; density: 0.910–0.935 g/cm3; oxygen transmittance: 4000 cm3/
m2 24 h. 0.1 MPa) and tightly sealed by a vacuum packaging machine (DZ-260, Bafang Mining Machinery Co., Ltd. Jining, China). The
sealed bags were then stored at 25 ± 0.5 ◦ C (room temperature) for 60 d.
2.3. Evaluations of alfalfa silage fermentation quality, chemical and microbial
At the end of the ensiling period, the bags were opened, and fresh sample of 20 g was collected from each bag and homogenized
with distilled water (180 mL) for 30 s in a juicer extractor (Zhang et al., 2020). Four layers of medical gauze were used to filter the
resulting mixture. Silage pH value was determined immediately from the filtrate by a portable pH meter. The filtrate was aliquoted into
three parts. The first part of the filtrate was stored at − 20 ◦ C to subsequently determine the antioxidant indexes. The second part of the
filtrate was acidified with 50% H2SO4 to achieve a pH = 2.0 and filtered through 0.22-μm membrane for the organic acid analysis as
described previously (Ding et al., 2013). A 25% (w/v) trichloroacetic acid (TCA) was added to the third portion of filtrate at a ratio of
4:1 (filtrate to TCA) and kept to stand at room temperature for 1 h to precipitate the true protein, the mixture was then centrifuged
(4 ◦ C, 10,000 × g, for 15 min) and the supernatant was stored for determining the contents of ammonia nitrogen (NH3-N) (Broderick
and Kang, 1980), non-protein nitrogen (NPN) (Abeysekara et al., 2013) and water-soluble carbohydrates (WSC) (Thomas, 1977).
Eighty grams of fresh forage or silage samples were dried at 65 ◦ C for 72 h in an air oven to determine the DM content. DM loss was
calculated after correcting the DM content according to the formula in the report of Porter et al. (1995). Thereafter, the dried samples
were ground and allowed to pass through a 1-mm sieve for analysis of crude protein (CP), neutral detergent fiber (aNDF) and acid
detergent fiber (ADF). Automatic Kjeldahl apparatus (K9840, Hannon instrument Co., Ltd. Jinan, China) was used to determine the
total nitrogen, and the CP content was calculated by multiplying the total nitrogen by 6.25 (AOAC, 1995). The contents of aNDF and
ADF were measured by the filter bag technique (ANKOM Technology, Fairport, NY, USA) (Van Soest et al., 1991). A heat stable
amylase and sodium sulfite were used during aNDF determination, and aNDF and ADF were expressed inclusive of residual ash.
To enumerate the microbial population in fresh material and silage, a 5 g fresh sample was taken from each bag and homogenized
with 50 mL sterilized physiological saline (8.5 g/L) in a constant temperature (37 ± 2 ◦ C) shaker for 20 min (120 RPM/min), and then
continuously diluted (10-folds). In total, a 100 µL from an appropriate dilution was spread on agar plates. The population of LAB was
counted on sterile-culture plates using MRS medium (37 ◦ C anaerobic culture for 48–72 h). The enumerations of yeasts and molds were
determined using Potato dextrose agar medium by incubation at room temperature for 5–7 d. An appropriately diluted plate with
colonies of 30–300 was used for the microbial enumerations (Reich and Kung, 2010).
2.4. Analysis of α-tocopherol, β-carotene, total flavonoid, lipoxygenase and fatty acid
The fresh alfalfa and silage were stored at − 80 ℃ refrigerators before freeze-dried using a lyophilizer (Bilon, Shanghai Billang
Instrument Manufacturing Co., Ltd, Shanghai, China) and ground by a precooled laboratory mill with a 1-mm screen for the analysis of
α-tocopherol, β-carotene, total flavonoid, lipoxygenase (LOX) and fatty acid. The concentrations of α-tocopherol and β-carotene were
obtained by an Agilent 7890A gas chromatograph (Agilent Technologies Inc., München, Germany) after saponification and extraction
according to the method of Zhang et al. (2020).
The total flavonoid concentration was determined based on a modified method of Singh et al. (2013). Briefly, a 0.2 g lyophilized
sample was weighed into a 15 mL glass tube and 10 mL methanol (60% w/v) was added. The mixture was vortexed for 30 s and
extracted for 24 h under dark conditions, and shaken every 8 h within the 24 h. The mixture was centrifuged for 10 min (3000 × g at
4 ◦ C), and the supernatant was used for the determination of total flavonoid. A 0.5 mL of the diluted supernatant (× 3) or 0.1, 0.2, 0.4,
0.6, 0.8, and 1 g/L standard solution of rutin (BW1681, Beijing Solarbio Science & Technology Co., Ltd, Beijing, China) was mixed with
0.3 mL NaNO2 (5% w/v) and kept for 5 min at room temperature. Subsequently, a 0.3 mL AlCl3 (10% w/v) solution was added and kept
for another 5 min at room temperature. A 2 mL NaOH (1 mol/L) was added and stored for last 10 min to terminate the reaction. The
absorbance of the reaction solution was determined by UV spectrophotometer at 510 nm (UV-3000PC, Mepoda-Technologies,
Shanghai, China). The total flavonoid content was expressed as rutin equivalent (RE, mg/g DM).
The LOX activity was determined following the description of Han and Zhou (2013) with some modifications. Briefly, a 1000 mg
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Animal Feed Science and Technology 288 (2022) 115301
X. Zhang et al.
lyophilized sample was diluted with 50 mmol/L phosphate buffer (pH = 7.0), and incubated on ice for 30 min with a vortex for 30 s
every 10 min. The incubated sample was centrifuged at 4 ◦ C; 10,000 × g for 30 min. The supernatant was used as the extract solution of
the LOX. A 0.2 mL supernatant was mixed with 1 mL linoleic acid substrate (250 μmol/L), and cultured at 30 ◦ C for 4 min. To terminate
the reaction, a 2.8 mL NaOH (1 mol/L) was added after cooling at room temperature. Meanwhile, a control test for each sample was
conducted, and the reaction time was placed at 0 min. Finally, the hydrogen peroxide produced by LOX was measured at 234 nm using
an ultraviolet spectrophotometer (UV-3000PC, Mepoda-Technologies, Shanghai, China). The LOX activity was calculated following
the formulas described previously (Zhang et al., 2021).
A modified method of O’Fallon et al. (2007) was used for the determination of fatty acid composition by gas chromatography-mass
spectrometry (GCMS-QP2010, MS Analysis and Test Technology Co. Ltd., Shimadzu, Japan) following fatty acid methyl ester (FAME)
synthesis. Briefly, a 0.5 g of lyophilized sample was extracted in a 10 mL glass tube with 6.3 mL methanol solution (0.1 mol/L) and 0.7
mL KOH (10 mol/L). The mixture was heated at 55 ◦ C for 90 min with a simple oscillation every 20 min. After the mixture was cooled
at room temperature, 0.58 mL of H2SO4 (12 mol/L) was added. Sample was reheated at 55 ◦ C for 90 min with a simple oscillation every
20 min. After cooling the sample again at room temperature, a 3 mL of internal standard solution [500 mg/L of methyl nonadecanoate
(C19:0)] was added. Finally, the mixture was centrifuged at 3000 rpm for 5 min after a thorough mixing. The supernatant was filtered
with 0.22-μm membrane, which was used to analyze fatty acid as described by Ke et al. (2017) using GC-MS fitted with a fused-silica
capillary column (100 m × 0.25 mm × 0.20 µm).
2.5. Enzyme activity analysis
The first filtrate previously stored at − 20 ◦ C was unfrozen and centrifuged at 4 ◦ C, 10,000 × g for 10 min. The T-AOC, SOD,
glutathione peroxidase (GSH-PX) and CAT activities were determined from the supernatant using assay kits (Nanjing Jiancheng
Bioengineering Research Institute Co., Ltd., Nanjing, China) according to the instruction manual.
2.6. Statistical analysis
The IBM Statistical Package for the Social Sciences (IBM SPSS 19.0 for Windows) was used for the statistical analysis. The data on
antioxidant properties of the screened strains (7 screened strains and control × 3 replicates) were analyzed by one-way analysis of
variance (ANOVA, General Linear Models). The data on fermentation quality, chemical and fatty acid compositions, and antioxidant
metabolites of silages (7 treatment and control × 4 replicates) according to a completely randomized design were analyzed to evaluate
the effect of the screened strains averaged across fields factor by one-way ANOVA. The means of different groups were then compared
for significance using Tukey’s test at P < 0.05. Trends were showed at 0.05 ≤ P ≤ 0.10. In addition, correlation between total
flavonoid, α-tocopherol, β-carotene and antioxidant enzyme was determined using Pearson correlation coefficient (PCCs) analysis.
3. Results
3.1. Antioxidant properties of screened strains
The screened strains, had strong free radical scavenging activities against DPPH, OH⋅, and O−2 , and with high antioxidant enzyme
activity (T-AOC, SOD) compared with commercial strain GFG (Table 1).
3.2. Effects of the screened strains on fermentation quality and microbial population
Ensiling process decreased (P < 0.001) pH value, but increased (P < 0.001) LAB number (Table 2). After ensiling, high pH value (>
5.0) was exhibited in commercial strain GFG-treated silage. Compared with control and commercial strain GFG-treated silages, in­
oculations of AS21, FM15, 13-7, and J17 increased (P < 0.001) lactic acid concentration. The highest lactic acid to acetic acid ratio (P
= 0.008) was in J17-treated silage. Higher butyric acid (P < 0.001) concentration was found in GFG-treated silage compared with
Table 1
Antioxidant properties of fermentation supernatant from lactic acid bacteria (n = 3).
Items*
GFG
24-7
BX62
AS21
FM15
13-7
J17
SEM**
P-value
T-AOC (U/mL)
SOD (U/mL)
GSH (μmol/L)
DPPH (%)
-OH⋅ (%)
O-2 (U/L)
8.84d
61.4b
29.6d
47.5e
53.8b
63.2d
10.3c
66.7a
33.0bc
50.4cd
65.9a
78.8a
12.7b
65.7a
34.2bc
57.5b
61.5ab
71.5b
13.7a
64.3a
36.3a
62.2a
69.3a
64.2d
10.3c
65.4a
33.8bc
48.6de
61.9ab
67.4c
10.7c
65.7a
34.4b
56.0b
64.5a
68.6c
10.7c
61.5b
32.7c
51.6c
64.2a
65.1d
0.25
0.65
0.52
0.88
1.06
0.95
< 0.001
< 0.001
< 0.001
< 0.001
< 0.001
< 0.001
GFG, Lactiplantibacillus plantarum GFG; 24-7, L. plantarum 24-7; BX62, L. plantarum BX62; AS21, L. plantarum AS21; FM15, L. plantarum FM15; 13-7,
Pediococcus acidilactici 13-7; J17, P. acidilactici J17.
*
T-AOC, total antioxidant capacity; SOD, superoxide dismutase; GSH, reduced glutathione; DPPH, 2, 2-diphenyl-1-picrylhydrazyl; -OH⋅, hydroxyl
radicals; O-2, superoxide anion.
**
SEM, standard error of the means.
4
Animal Feed Science and Technology 288 (2022) 115301
X. Zhang et al.
Table 2
pH, organic acid contents and microbial population of alfalfa silages ensiled for 60 d (n = 4).
Item*
Fresh alfalfa
Control
GFG
24-7
BX62
AS21
FM15
13-7
J17
SEM**
P-value
pH
Lactic acid, g/kg DM
Acetic acid, g/kg DM
Propionic acid, g/kg DM
Lactic acid/Acetic acid
Butyric acid, g/kg DM
LAB (log10 CFU/g FW)
Yeasts (log10 CFU/g FW)
Molds (log10 CFU/g FW)
6.04a
–
–
–
–
–
6.30d
6.35
6.41
4.86d
69.2b
28.1bc
14.6cd
2.46bc
0.51b
7.72ab
ND
ND
5.12b
65.6b
35.2a
15.7bc
1.86c
0.74a
7.69ab
ND
ND
4.89d
67.0b
30.7a–c
17.8ab
2.18c
0.46b
7.59b
ND
ND
4.95c
67.2b
32.3ab
15.1bc
2.08c
0.51b
7.91a
ND
ND
4.67e
89.4a
31.2ab
19.1a
2.87b
0.44b
7.45bc
ND
ND
4.69e
86.4a
27.7bc
17.3a–c
3.12b
0.41b
7.45bc
ND
ND
4.54f
91.6a
28.8a–c
16.3a–c
3.18b
0.41b
7.47bc
ND
ND
4.55f
93.7a
23.6c
14.4cd
3.98a
0.39b
7.52b
ND
ND
0.031
2.56
1.19
0.51
0.164
0.002
0.418
–
–
< 0.001
0.001
0.002
< 0.001
< 0.001
< 0.001
< 0.001
–
–
a–e
Means with different superscript letters within a row significantly differ (P < 0.05).
GFG, Lactiplantibacillus plantarum GFG; 24-7, L. plantarum 24-7; BX62, L. plantarum BX62; AS21, L. plantarum AS21; FM15, L. plantarum FM15; 13-7,
Pediococcus acidilactici 13-7; J17, P. acidilactici J17.
ND, less than 5.47 log10 CFU/g FW, and not counted.
*
LAB, lactic acid bacteria; CFU, colony forming unit; DM, dry matter; FW, fresh weight.
**
SEM, standard error of the means.
other silages. Higher acetic acid concentration was observed in GFG-treated silage versus control (P = 0.017), FM15- (P = 0.03), and
J17-treated (P = 0.002) silages.
3.3. Chemical composition, α-tocopherol, β-carotene and total flavonoid concentrations in fresh alfalfa and silages
Increased NPN (72.2%) and NH3-N (92.1%) concentrations as well as decreased WSC (85.8%) content were observed after natural
ensiling (Table 3). Compared with control and commercial strain GFG-treated silages, AS21- (P < 0.001), FM15- (P = 0.007), 13-7- (P
= 0.03), and J17-treated (P < 0.001) silages decreased NH3-N concentration. No difference in NPN concentration of alfalfa silage was
observed after inoculating with the screened strains. AS21-treated silage had a lower (P = 0.04) WSC concentration versus control.
Additionally, after ensiling, the highest DM loss (P = 0.003) was in GFG-treated silage.
Lower α-tocopherol (65.9 mg/kg, P < 0.001) and β-carotene (41.4 mg/kg, P < 0.001) concentrations were found after natural
ensiling (Table 3). Compared with control and commercial strain GFG-treated silages, all inoculants with high-antioxidant activity
reduced the α-tocopherol (P < 0.001) and β-carotene (P < 0.001) losses. The highest α-tocopherol concentration (P = 0.003) was
observed in BX62 inoculation among silages treated with antioxidant strains. The highest β-carotene concentration (P < 0.001) was
observed in silages inoculated with 24-7, BX62, AS21 and J17 strains. Increase of total flavonoid (11.8%) concentration was observed
after natural ensiling. Higher total flavonoid concentration was found in 24-7- (P = 0.004), BX62- (P < 0.001), FM15- (P = 0.03), J17treated (P < 0.001) silages compared with control.
3.4. Fatty acid composition in fresh alfalfa and silages
The main fatty acids in fresh alfalfa were C16:0, c9,12,15C18:3 and c9,12C18:2 (Table 4). The C14:0 (P = 0.002), C16:0 (P =
Table 3
Chemical composition, α-tocopherol, β-carotene and total flavonoid concentrations of fresh alfalfa and alfalfa silages ensiled for 60 d (n = 4).
Item*
Fresh
alfalfa
Control
GFG
24-7
BX62
AS21
FM15
13-7
J17
SEM
**
P-value
DM, g/kg FW
DM loss, g/kg FW
WSC, g/kg DM
CP, g/kg DM
NPN, g/kg TN
NH3-N, g/kg TN
aNDF, g/kg DM
ADF, g/kg DM
α-tocopherol, mg/kg DM
β-carotene, mg/kg DM
Total flavonoid (mg of rutin
equivalent/g of DM)
437a
–
53.2a
225
289b
4.56f
358
242
174a
88.0a
30.6e
426a–c
76.2b
7.55bc
235
430a
57.6b
380
256
65.9i
41.4f
34.6cd
423a–c
82.5a
7.97b
219
495a
69.4a
386
263
73.9h
62.6e
31.8d
417bc
67.8c
7.53bc
238
452a
54.6b
376
246
85.9d
78.8b
39.5a
421a–c
62.4c
8.26b
224
486a
51.4bc
361
230
97.6b
79.2b
41.5a
418a–c
62.1c
5.19d
239
426a
37.4d
364
237
84.3e
78.3b
35.6bc
414c
59.3cd
6.18b–d
225
432a
46.2c
369
248
78.8f
74.5c
38.7ab
436a
43.2d
7.99b
219
451a
36.9d
335
252
75.6g
70.1d
35.9bc
422a–c
45.3d
7.81b
236
454a
25.5e
350
236
87.4c
78.4b
41.6a
1.7
1.05
0.208
2.3
3.0
1.57
7.9
3.4
1.87
2.18
0.45
0.005
< 0.001
< 0.001
0.324
0.045
< 0.001
0.648
0.218
< 0.001
< 0.001
< 0.001
a–i
Means with different superscript letters within a row significantly differ (P < 0.05).
GFG, Lactiplantibacillus plantarum GFG; 24-7, L. plantarum 24-7; BX62, L. plantarum BX62; AS21, L. plantarum AS21; FM15, L. plantarum FM15; 13-7,
Pediococcus acidilactici 13-7; J17, P. acidilactici J17.
*
CFU, colony forming unit; FW, fresh weight; DM, dry matter; WSC, water soluble carbohydrates; CP, crude protein; NPN, non-protein nitrogen;
NH3-N, ammonia nitrogen; TN, total nitrogen, aNDF, neutral detergent fiber; ADF, acid detergent fiber.
**
SEM, standard error of the means.
5
Animal Feed Science and Technology 288 (2022) 115301
X. Zhang et al.
0.003), C18:0 (P < 0.001), C20:0 (P = 0.025), and C22:0 (P < 0.001) increased in control silage relative to fresh alfalfa. The lowest
proportion of saturated fatty acid (SFA) (P < 0.001) was in 24-7-treated silage. In comparison to control and GFG-treated silages, 24-7(P < 0.001), BX62- (P < 0.001), AS21- (P = 0.004), FM15- (P = 0.002), J17-treated (P < 0.001) silages had higher proportion of
polyunsaturated fatty acid (PUFA). Proportion of c9,12C18:2 increased (P < 0.001) in silages inoculated with screened strains
compared with control and GFG-treated silages. The highest proportion of c9,12,15C18:3 (α-linolenic acid) (P < 0.001) was found in
BX62-treated silage among the screened strains-treated silages.
3.5. Change of antioxidant enzyme and lipoxygenase activities in silages
Except for strain J17, inoculations with the screened strains had higher (P < 0.001) T-AOC activity in alfalfa silage compared with
control and GFG-treated silages (Table 5). The highest T-AOC (200 U/g, P < 0.001) activity was in AS21-treated silage. Inoculated
silages had higher (P < 0.001) GSH-PX activity versus control. Compared with the control, higher CAT concentration was in 24-7- (P =
0.002), BX62- (P < 0.001), AS21- (P = 0.03), J17-treated (P < 0.001) silages. Conversely, compared with control and commercial
strain GFG-treated silages, lower SOD activity was observed in FM15-(P < 0.001) and J17-treated (P < 0.001) silages. Lower LOX
activity (P < 0.001) was observed in silages inoculated with the screened strains compared with control and GFG-treated silages.
4. Discussion
4.1. Effect of antioxidant strains on fermentation quality of silages
The natural ensiling and LAB inoculation are able to produce sufficient lactic acid concentration to decrease pH and inhibit un­
desirable bacteria (Lima et al., 2010; Guan et al., 2016). This was consistent with the present result, which neither mold nor yeast was
found in inoculants-treated silages and control silage under the dilution ratio used in this experiment. Unexpectedly, GFG-treated
silage had poor fermentation demonstrated by higher pH value and butyrate concentration, lower lactic acid concentration, and se­
vere DM loss. One possible reason was that commercial strain GFG inoculation inhibited proportion of beneficial microbes under
present fermentation conditions. Oliveira et al. (2017) reported that lower lactic acid concentration could be obtained in the presence
of some inoculants. It also may be that secondary fermentation occurred during the fermentation process, resulting in more nutrients
loss (Wu et al., 2021). In addition, higher lactic acid concentration in AS21-, FM15-, 13-7- and J17-treated silages was detected
compared with control and commercial strain GFG-treated silages. The reason for this may be that inoculations of these screened
strains can facilitate lactic acid fermentation during ensiling.
4.2. Effect of antioxidant strains on chemical composition, α-tocopherol, β-carotene and total flavonoid concentrations in silages
Compared with the control, screened strains inoculants were able to preserve more WSC concentration in alfalfa silage due to the
decrease in nutrients losses caused by undesirable bacteria (Sun et al., 2009; Guo et al., 2014). However, AS21-treated silage had a
lower WSC concentration versus control, which could be attributed to the utilization of large amounts of sugar by the enzymes (Hu
et al., 2009). The concentrations of NPN and NH3-N increased after natural ensiling, indicating protein was hydrolyzed to NPN, NH3-N,
and so forth. In the present study, AS21, FM15, 13-7 and J17-treated silages had lower NH3-N concentration compared with the
Table 4
Total fatty acid content (g/kg of DM) and the major fatty acid compositions (g/100 g of Total fatty acid) of fresh alfalfa and alfalfa silage ensiled for 60
d (n = 4).
Item*
Fresh alfalfa
Control
GFG
24-7
BX62
AS21
FM15
13-7
J17
SEM**
P-value
TFA(g/kg)
C14:0
C16:0
C18:0
t9C18:1
c9C18:1
c11,14C18:2
c9,12C18:2
c9,12,15C18:3
C20:0
C22:0
C24:0
SFA
MUFA
PUFA
16.4a
4.73d
24.7d
6.65e
1.94bc
0.57a
1.22a
15.6a
41.2a
1.23d
1.59cd
2.46bc
43.3d
3.09ab
58.0a
13.4b
5.02bc
27.2c
7.40a
2.05a–c
0.53a
0d
10.4e
31.5ef
1.52ab
1.75ab
2.31c
48.3ab
3.14ab
41.9e
12.8b–d
5.55a–c
28.4bc
7.05d
2.42a
0.35b
0.04d
10.6e
28.8g
1.43bc
1.82a
2.43bc
48.1ab
3.33a
39.4f
12.5c–e
5.24a–c
27.3c
7.32ab
1.87c
0.22c
0.86b
13.1bc
33.9c
1.31c
1.70bc
2.61ab
45.5c
2.64c
47.8bc
12.7cde
5.79ab
28.8abc
7.17bcd
2.20abc
0.19c
0.68bc
13.5b
34.9b
1.53ab
1.71a–c
2.52bc
47.5a–c
2.95a–c
49.0b
13.1bc
4.99c
28.6a–c
7.09cd
2.03bc
0.23c
0.37cd
13.2b
33.5c
1.59ab
1.61c
2.73a
46.6bc
2.82bc
47.1c
12.5c–e
5.08bc
27.6c
7.21a–d
2.28ab
0.11d
0.26d
12.11cd
32.2d
1.64a
1.81ab
2.79a
46.1bc
2.95abc
44.6d
12.1e
5.97ab
29.8a
7.38ab
1.97bc
0.22c
0.10d
11.8d
31.1f
1.58ab
1.77ab
2.44bc
49.0a
2.75bc
42.4e
12.4de
5.58a–c
30.1a
7.28a–c
1.99bc
0.21c
0.11d
12.2cd
33.3c
1.65a
1.80ab
2.43bc
48.9a
2.76bc
45.6d
0.08
0.076
0.21
0.031
0.040
0.022
0.057
0.22
0.33
0.017
0.009
0.045
0.27
0.045
0.56
< 0.001
0.002
< 0.001
< 0.001
0.003
< 0.001
< 0.001
< 0.001
< 0.001
< 0.001
< 0.001
< 0.001
< 0.001
< 0.001
< 0.001
a–f
Means with different superscript letters within a row significantly differ (P < 0.05).
GFG, Lactiplantibacillus plantarum GFG; 24-7, L. plantarum 24-7; BX62, L. plantarum BX62; AS21, L. plantarum AS21; FM15, L. plantarum FM15; 13-7,
Pediococcus acidilactici 13-7; J17, P. acidilactici J17.
*
TFA, total fatty acids; SFA, saturated fatty acid; MUFA, monounsaturated fatty acid; PUFA, polyunsaturated fatty acid.
**
SEM, standard error of the means.
6
Animal Feed Science and Technology 288 (2022) 115301
X. Zhang et al.
Table 5
Antioxidant enzyme and lipoxygenase activities of alfalfa silages ensiled for 60 d (n = 4).
Item*
Control
GFG
24-7
BX62
AS21
FM15
13-7
J17
SEM**
P-value
T-AOC, U/g FW
SOD, U/g FW
CAT, U/g FW
GSH-PX, U/g FW
LOX, U/g FW
147e
576ab
11.9e
1530d
532a
159cd
592a
14.2b–e
1649bc
612a
177b
563a–c
15.7a–d
1757ab
0d
180b
573ab
16.1a–c
1744ab
0d
200a
587ab
15.2a–d
1639c
23.1c
173b
544c
14.1c–e
1681a–c
16.3c
173b
551bc
12.9de
1654bc
97.4b
167bc
509d
17.0ab
1719a–c
0e
2.2
4.5
0.37
13.7
0.67
< 0.001
< 0.001
< 0.001
< 0.001
< 0.001
a–e
Means with different superscript letters within a row significantly differ (P < 0.05).
GFG, Lactiplantibacillus plantarum GFG; 24-7, L. plantarum 24-7; BX62, L. plantarum BX62; AS21, L. plantarum AS21; FM15, L. plantarum FM15; 13-7,
Pediococcus acidilactici 13-7; J17, P. acidilactici J17.
*
T-AOC, total antioxidant capacity; SOD, superoxide dismutase; GSH-PX, glutathione peroxidase; CAT, catalase; LOX, lipoxygenase; FW, fresh
weight.
**
SEM, standard error of the means.
control. The reason could be that these LAB had inhibited the activity of proteolytic enzymes during ensiling (Ke et al., 2017). Zhang
et al. (2021) also showed that inoculation of L. plantarum 24-7 with high-antioxidant capacity reduced NH3-N concentration in alfalfa
silage.
The breakdowns of α-tocopherol and β-carotene were attributed to microbes and lipid-related enzymes (Elgersma et al., 2013; Wu
et al., 2021). Interestingly, inoculations of any of the six screened strains could effectively inhibit the degradations of α-tocopherol and
β-carotene in ensiled alfalfa. The reason may be that the inoculations of screened strains with antioxidant activity inhibit microbes and
lipid-related enzymes associated with α-tocopherol and β-carotene degradations in alfalfa silage (Wu et al., 2021). Similarly, our
previous studies also reported that α-tocopherol and β-carotene concentrations in alfalfa silage could be improved by inoculation of
LAB with antioxidant activity (Zhang et al., 2020; Zhang et al., 2021).
As a polyphenol, flavonoids possess high antioxidant activity, which may attribute to their polyhydroxy structure (Iqbal et al.,
2012). This special structure can inhibit the activity of enzymes that catalyzes the O-2 production (Bao et al., 2016). Increase of total
flavonoid (11.8%) concentration was observed after natural ensiling. Higher total flavonoid concentration was found in 24-7-, BX62-,
FM15-, J17-treated silages compared with the control. The reason for this was that inoculations of these screened strains conduced to
improve antioxidant activity in alfalfa silage by increasing flavanol biosynthesis. As it was reported, L. buchneri inoculation in corn
silage upregulated the flavones and flavanol biosynthesis pathways (Xu et al., 2020).
4.3. Effect of antioxidant strains on fatty acid composition in silages
The fatty acid composition of silage has attracted considerable attention because high intake of PUFA is hoped to increase the PUFA
concentration in ruminant products (Lee et al., 2008), and consequently may be beneficial to human health (Kaushik et al., 2019).
However, ensiling normally causes loss of forage PUFA (Alves et al., 2011). Compared with the control, LAB-treated silages showed
higher proportion of PUFA except for GFG and 13-7-treated silages. In particular, we found that 24-7-, BX62-, AS21-, FM15- and
J17-treated silages had higher proportion of α-linolenic acid compared with the control. These findings demonstrated that most
screened strains with high-antioxidant activity could inhibit the biohydrogenation or lipid peroxidation of unsaturated fatty acid
Table 6
Pearson correlations between antioxidant activities (T-AOC, SOD, CAT, GSH-PX and LOX) and total flavonoid, α-tocopherol, β-carotene of alfalfa
ensiled for 60 d.
Itema
T-AOC
SOD
T-AOC
1
0.15
0.71
1
SOD
CAT
GSH-PX
CAT
0.17
0.66
-0.38
0.31
1
GSH-PX
LOX
Total flavonoid
α-tocopherol
β-carotene
0.20
0.62
-0.40
0.29
0.87*
0.002
1
-0.67*
0.05
0.36
0.34
-0.65
0.06
-0.79*
0.01
1
0.40
0.29
-0.55
0.12
0.35
0.36
0.43
0.25
-0.51
0.17
1
0.66*
0.05
-0.18
0.65
0.61
0.08
0.64
0.07
-0.74*
0.02
0.79*
0.01
1
0.76*
0.02
-0.32
0.40
0.64
0.06
0.74*
0.02
-0.98*
< 0.001
0.62
0.08
0.84*
0.01
1
LOX
Total flavonoid
α-tocopherol
β-carotene
Upper values for each category denote correlation coefficients; lower values denote P-value of the correlation; P-value ≤ 0.05 as the correlation
standard; “*” show significant correlation coefficients; “+” indicates positive correlation, and “-” indicates negative correlation.
a
T-AOC, total antioxidant capacity; SOD, superoxide dismutase; GSH-PX, glutathione peroxidase; CAT, catalase; LOX, lipoxygenase.
7
Animal Feed Science and Technology 288 (2022) 115301
X. Zhang et al.
during ensiling. Van Ranst et al. (2009) reported that a high biohydrogenation rate was observed in ensiling process because most of
the fatty acid in fresh forages exist in the free fatty acid form. Previous studies have also shown that the inoculations of some LAB can
reduce the PUFA hydrogenation (Zhang et al., 2020, 2021).
Lipoxygenase from the plant and microbes is generally considered as an oxidoreductase, which is involved in the oxidation process
of unsaturated fatty acids and degradation of antioxidant metabolites (Yang and Koo, 2000; Wu et al., 2021). The lowered LOX activity
in the screened strains-treated silages was detected compared with control and commercial strain GFG-treated silages. This was
attributed to the ability of the screened strains-treated silages to inhibit the oxidation process of unsaturated fatty acids by lip­
oxygenase (Han and Zhou, 2013). As shown in Table 6, pearson correlations analysis also indicated that LOX was negatively correlated
with α-tocopherol and β-carotene concentrations, and T-AOC activity. In addition, the reason that LOX activity was not affected in
GFG-treated silage might be high pH value (> 5) in GFG-treated silage. Lourenço et al. (2005) showed LOX activity would be inhibited
by low pH value.
4.4. Changes of antioxidant enzyme activity in silages ensiled with screened strains
Oxidative stress in animals has a negative impact on animal production and the products quality as well (Miller et al., 1993). The
LAB have strong antioxidant activities following production of antioxidant enzymes (such as SOD, GSH-PX, CAT and T-AOC) and
non-enzymatic antioxidants (including vitamin E, carotene and flavonoids, etc.) (Kenfack et al., 2018). In the present study, T-AOC,
GSH-PX and CAT activities were higher in most screened strains treated-silages compared with control and GFG treated-silages, which
suggested that not all antioxidant LAB could improve the antioxidant status of ensiled forage by antioxidant enzymes. Generally, the
SOD is the first line of defense against oxidation, and plays significant role in free radicals’ scavenging. The SOD can transport O-2
generated in an electron transfer reaction by transporting oxygen O2 in alkaline and anaerobic environment (Hyland et al., 1983).
Present results showed that FM15- and J17-treated silages had lower SOD activity compared with control and commercial strain
GFG-treated silages. One of the possible explanations is that the aerobic pathway will be shut down in an anaerobic fermentation
process, and resulting in a rapid decline of pH in the presence of LAB. Therefore, the inoculations of FM15 and J17 strains in alfalfa
silage reduced the generation of O-2 in low pH value and led to lower SOD activity compared with control and commercial strain GFG
treated-silages.
Under present situation, the results suggested that all the tested LAB strains with high antioxidant activities could be used as
candidate strains to improve antioxidant status and fermentation quality of ensiled alfalfa, and strain AS21 was the most preferred
strain. The different antioxidant strains may have different antioxidant mechanisms during ensiling. However, it is noteworthy that the
present results may not be reproducible if alfalfa silage inoculated with the screened strains with high antioxidants are to be made in
larger mini-silos. The mini-silos removal of high volume of air at ensiling could have influence the results, which is in relation with the
implication it has on the ensiling process and the different antioxidant. Therefore, in future experiments, it will be preferred to use
larger mini-silos allowing longer contact with oxygen after sealing, for examples, 4–5 kg buckets at a density of around 200 kg per
cubic meter.
5. Conclusion
Application of the screened LAB strains with high-antioxidant activity improved alfalfa silage fermentation quality, with a better
fermentation quality being observed in silage treated with AS21, FM15, 13-7, and J17 strains. Inoculations of the screened LAB
reduced the α-tocopherol and β-carotene losses. Most of the antioxidant strains used in the present study improved T-AOC activity of
alfalfa silage, and the highest T-AOC was observed in AS21-treated silage. Therefore, all the screened LAB strains with high-antioxidant
activity tested in the present study could be used as candidate strains to improve antioxidant status and fermentation quality of ensiled
alfalfa, with AS21 strain as the most preferred strain.
CRediT authorship contribution statement
X. Zhang: Methodology, Validation, Investigation, Formal analysis, Writing – original draft. X.S. Guo: Conceptualization, Su­
pervision, Writing – review & editing, Project administration, Funding acquisition. F.H. Li: Validation, Investigation, Formal analysis.
S. Usman: Grammar, Validation, Investigation. Y.X. Zhang: Methodology, Validation, Investigation. Z.T. Ding: Supervision, Writing
– review & editing, Project administration, Funding acquisition.
Declaration of Competing Interest
The authors declare that they have no known competing for financial interests or personal relationships that could have appeared to
influence the work reported in this paper.
Acknowledgement
This work was supported by the National Natural Science Foundation of China, China (Grant No. 31901390).
8
Animal Feed Science and Technology 288 (2022) 115301
X. Zhang et al.
Appendix A. Supporting information
Supplementary data associated with this article can be found in the online version at doi:10.1016/j.anifeedsci.2022.115301.
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