Effects of Lactic Acid Bacteria Additives on Fatty Acids, Amino Acids and Antioxidant Capacity of Leymus chinensis Silage during Aerobic Exposure

Abstract

During aerobic exposure of silage, the fatty acid and amino acid composition may alter the quality and palatability, resulting in economic losses in livestock production. The objective of this study was to evaluate the effects of Lactiplantibacillus plantarum (LP), Lenti Lentilactobacillus buchneri (LB), and a mixture of LP and LB (PB) on the fatty acids, amino acids, and antioxidant capacity of Leymus chinensis silage during aerobic exposure. The lactic acid bacteria were added at 1 × 10⁶ CFU/g. The silage treatments were opened after 60 days of fermentation, and sampled on days 0, 4, and 8 of aerobic exposure. The LB group had higher total fatty acid and polyunsaturated fatty acid content, and less decrease in amino acid content and antioxidant capacity, while the LP group had a higher monounsaturated fatty acid content but a larger decrease in all indicators after exposure. Correlation analysis showed that Lactobacillus, Cryptococcus, Penicillium, and Thermoascus were more correlated with fatty acid changes, and that Lactobacillus, Actinomyces, Clostridium, and Penicillium were more correlated with amino acid changes. In conclusion, Lentilactobacillus buchneri could effectively improve the antioxidant capacity and fatty acid and amino acid contents of Leymus chinensis silage during aerobic exposure, while Lactiplantibacillus plantarum could effectively improve the content of each index of Leymus chinensis silage at opening, but deterioration was faster during aerobic exposure.

1. Introduction

Leymus chinensis is widely distributed in the northeastern plains of China and the eastern part of the Inner Mongolia plateau, and in recent years the use of Leymus chinensis silage has gradually increased due to the effect of rainfall on hay drying [1]. Silage is the main plant food in ruminant rations, and Leymus chinensis silage is rich in fatty acids and amino acids, but the oxidation and reproduction of aerobic microorganisms during aerobic exposure can affect the composition of fatty acids and amino acids. Fatty acids are primarily divided into saturated and unsaturated fatty acids, and the unsaturated fatty acids are further broken down into monounsaturated fatty acids and polyunsaturated fatty acids [2]. Monounsaturated fatty acids in forage include oleic acid and others, and polyunsaturated fatty acids include linoleic acid, α-linolenic acid, and so forth [3,4]. Feeds rich in unsaturated fatty acids are readily oxidized and then undergo oxidative decomposition, polymerization, and other reactions, leading to rancidity and even toxicity, all of which can cause harm to livestock performance. A large body of research has demonstrated that the type, content, and distribution of unsaturated fatty acids in milk and meat can be altered by animal feed [5,6].

Some studies have reported that peroxides generated from the oxidation of unsaturated fatty acids can react with many active amino acid residues in protein molecules, especially amino acids containing sulfur, which can lead to aggregation of proteins, reduced solubility or enzymatic properties, or a decrease in nutritional value [7]. During fatty acid oxidation, the methionine residues in proteins are also oxidized to methionine sulfide or sulfone, and both cysteine and cystine are oxidized to monoxides or dioxides [8]. During aerobic exposure, amino acids undergo catabolism by first removing the amino groups, and the resulting carbon backbone can be oxidized to CO₂ and H₂O [9]. Concurrently, the amino acid content of spoiled silage decreases as the volume of spoilage increases, and amino acid loss is associated with a corresponding increase in the content nitrogen containing compounds such as ammonia nitrogen, which could be due to the oxidation of amino acids and metabolism of aerobic and fungal growth [10]. Many studies have shown that the use of additives can inhibit the growth of aerobic bacteria and enhance aerobic stability [11], and feeds with a high capacity for antioxidants can effectively enhance the yield of livestock [12]. The addition of Lactiplantibacillus plantarum to silage produces volatile fatty acids and essential amino acids, and Lentilactobacillus buchneri produces specific amino acids [13]. However, there has been little research into the effects of additives on antioxidant capacity, fatty acid, and amino acid oxidation processes during aerobic exposure to air of silage.

Aerobic spoilage severely affects silage quality and result in severe economic losses to the development of animal husbandry [14,15]. Improving the aerobic stability and antioxidant capacity of silage can effectively reduce nutrient loss and toxin production during aerobic exposure of silage, ensuring the safety of livestock feed. Meanwhile, reducing the oxidative decomposition of fatty acids and amino acids can avoid the production of poor odor and taste, and improve the intake of livestock and the palatability of feed [16,17]. The purpose of this study was to investigate the effects of lactic acid bacteria additives on fatty acids, amino acids, and antioxidant capacity of Leymus chinensis silage during aerobic exposure, and to analyze changes and correlations to provide evidence to ensure the quality of Leymus chinensis silage during aerobic exposure.

2. Materials and Methods

2.1. Silage Preparation

The variety of Leymus chinensis used in this experiment was Jisheng No. 1, harvested at the Science and Technology Park of Inner Mongolia University for Nationalities, Tongliao City, Inner Mongolia Autonomous Region (E43°59′, N122°11′). The Leymus chinensis was harvested at the tassel stage, laid flat for 3 h, then chopped to 2 cm with a guillotine (Shandong Jufeng Hardware Cutting Tools Factory, Linyi, China) and mixed evenly. Fresh samples were stored in a −80 °C refrigerator for determination of raw material characteristics. The treatment settings for Leymus chinensis silage were as follows: uninoculated control group (CK), Lactiplantibacillus plantarum group (LP), Lentilactobacillus buchneri group (LB), a mixture of LP and LB group (PB, 1:1). The amount of Lactobacillus added was 1 × 10⁶ CFU/g (all provided by Shandong Zhongke Jiayi Biological Engineering Co., Ltd., Weifang, China), and the same amount of distilled water was added to the control group. Samples of 300 g were weighed and mixed into silage bags (25 cm × 36 cm) and then vacuum sealed. Three replicates were set up for each treatment and these were stored at room temperature and shielded from light (20–30 °C) for 60 days.

2.2. Aerobic Stability

After 60 days of ensiling, the samples were opened and transferred into sterile bottles. Then multichannel data loggers (Model: MDL-1048A; Shanghai Tianhe Automation Instruments Co., Ltd., Shanghai, China) were inserted into the bottles to monitor changes in the silage temperature, and three probes were used to monitor changes in the ambient temperature. The mouth of the sterile bottles was wrapped with sterile gauze to reduce contamination and loss of moisture. This experiment was designed to determine the time required for the aerobically stable silage temperature to rise above room temperature by more than 2 °C [18].

2.3. Antioxidant Capacity

Samples of silage exposed to oxygen for 0, 4, and 8 days were crushed in a mortar, and a 0.5 g sample was placed in a stoppered conical flask. Then, 5 mL of extraction solvent (80% ethanol solution) was added and the samples were soaked at room temperature for 5 h with intermittent shaking. The extract was centrifuged at 3500 r/min for 10 min, the precipitate was discarded, and the supernatant was diluted to 5 mL. Two milliliters of extract were pipetted into a 10 mL test tube with a stopper and 2 mL of DPPH (2,2-Diphenyl-1-picrylhydrazyl) solution was added and mixed well. The absorbance Ai was measured by spectrophotometer at 517 nm after 30 min of standing at room temperature (80% ethanol solution was adjusted to zero). The absorbance Ac of 2 mL of DPPH solution mixed with 2 mL of 80% ethanol solution and the absorbance Aj of 2 mL of extract mixed with 2 mL of 80% ethanol solution were measured simultaneously. The inhibition rate was calculated by the following equation after taking the average of three parallel measurements. The greater the inhibition rate, the stronger the antioxidant activity of the sample.

$$X(\%)=(1-\frac{Ai-Aj}{Ac})\times 100$$

(1)

A 20 mg sample was weighed, dissolved by adding 95% ethanol and diluted to 2 mL to obtain 1 mg/mL sample solution. Then, 1 mL of 2 mol/L Folin–Ciocalteu reagent and 7 mL of distilled water were added, and the solution was stirred thoroughly to obtain 0.25 mol/L of Folin–Ciocalteu reagent. Next, 3.0 g Na₂CO₃: 10H₂O was dissolved in 20 mL of water and mixed to obtain a solution of 15% Na₂CO₃. Then, 0.5 mL of the sample solution and 0.5 mL of 0.25 mol/L Folin–Ciocalteu reagent were mixed, left to stand for 3 min before 1 mL of 15% Na₂CO₃ solution was added. The mixture was then left to stand for 30 min, centrifuged at 3500 r/min for 3 min, and the absorbance A value at 760 nm was measured. The total phenol concentration C was obtained by substituting into the standard curve [19,20].

$$A=32.14527\times C+0.03271 R=0.999$$

(2)

2.4. Fatty Acid

Gas chromatography was used to determine the fatty acids in Leymus chinensis silage. Petroleum ether was added to the sample for extraction, and then the solvent was removed by distillation followed by drying. Then, a 50 mg sample was taken to which was added 4 mL of hexane and 75 mg of anhydrous sodium sulfate, followed by stirring. Then, 0.20 mL of potassium methoxide and 2 mL of hydrochloric acid solution were added with constant stirring and heating for 20 min. After stirring the mixture several times, the supernatant containing the fatty acid methyl ester was extracted for analysis by gas chromatography (model: GC-2010plus, Shimadzu, Kyoto, Japan).

2.5. Amino Acid

After drying, grinding and sieving with 100 mesh sieve, 0.5 mg of dry sample was weighed into a 20 mL anaerobic test tube. Then, 10 mL of 6 mol/L HCL was added, nitrogen was applied for 30 s, and the sample was then placed in a 110 °C furnace for 24 h hydrolysis. The samples were cooled down, mixed well, filtered and made up to 25 mL in a volumetric flask with ultrapure water. Then, 1 mL of the filtrate was introduced into a beaker, heated in a water bath at 60 °C, evaporated to dryness, and finally, 5 mL of sample dilution solution was added to obtain the appropriate amino acid concentration. The solution to be measured was shaken and mixed and then filtered using a disposable syringe with a 0.22 μm filter, before being injected into the injection vial for measurement by the machine. The instrument is based on the S-433D amino acid analyzer model, and the production company is SYKAM in Germany. This instrument uses the classic method of ninhydrin post-column derivatization to analyze amino acids in hydrolyzed proteins and physiologic fluids, and can detect qualitatively and quantitatively all currently relevant amino acids.

2.6. Statistical Analysis

One-way ANOVA and Dunnett’s multiple comparisons were performed on the measured silage indicators using the statistical package SAS 9.2, and the level of p < 0.05 was considered statistically significant. Correlation coefficients between each indicator and the microbial community were calculated using the Pearson method (Supplementary Materials Table S1), and data on microbial diversity were taken from previous experiments [21]. Tables were produced using Excel 2010 and images were generated using GraphPad prism 8.0.2 and R version 3.6.3.

3. Results

3.1. Silage Raw Material Characteristics

The fatty acids, amino acids, and antioxidant capacity of the silage raw material are shown in

Table 1. Fatty acids, amino acids and antioxidant capacity of raw materials.

	Items	Sample	SEM
Fatty Acids (%)	TFA	1.4839	0.0120
	SFA	0.4843	0.0056
	PUFA	0.7044	0.0179
	MUFA	0.2952	0.0022
	C14:0	0.0509	0.0005
	C15:0	0.0056	0.0000
	C16:0	0.3602	0.0055
	C16:1	0.0423	0.0004
	C17:0	0.0048	0.0003
	C18:0	0.0433	0.0008
	C18:1n9t	0.0245	0.0001
	C18:1n9c	0.2284	0.0018
	C18:2n6c	0.2329	0.0045
	C20:0	0.0161	0.0003
	C18:3n3	0.4715	0.0222
	C22:0	0.0000	0.0000
	C20:3n6	0.0000	0.0000
	C24:0	0.0035	0.0003
Amino Acids (%)	TAA	12.9613	0.0154
	EAA	6.1690	0.0570
	NEAA	6.7923	0.0426
	Arg *	0.7243	0.0146
	His *	0.4057	0.0215
	Ile *	0.5631	0.0109
	Leu *	1.1381	0.0260
	Lys *	0.7260	0.0116
	Met *	0.1451	0.0021
	Phe *	0.7991	0.0116
	Thr *	0.6157	0.0184
	Val *	1.0517	0.0105
	Ala	1.1814	0.0217
	Asp	1.7258	0.0614
	Cys	0.0894	0.0018
	Glu	1.4910	0.0425
	Gly	0.6557	0.0145
	Pro	0.6928	0.0085
	Ser	0.5414	0.0105
	Tyr	0.4147	0.0074
Antioxidant capacity (%)	Total Phenol	0.3732	0.0146
	Inhibition Rate	72.9067	0.4491

SEM represents the standard error of the mean, * represents non-essential amino acids.

. The contents of TFA, SFA, PUFA, and MUFA were 1.4839%, 0.4843%, 0.7044%, and 0.2952%, respectively. The contents of TAA, EAA, and NEFA were 12.9613%, 6.1690%, and 6.7923%, respectively. The total phenol content of fresh Leymus chinensis was 1.4262%, and the free radical inhibition rate was 82.4578%. The SEM value is small and the differences within the group are small.

3.2. Temperature Difference between Silage and Room Temperature during Aerobic Exposure

Figure 1 shows the difference between the Leymus chinensis silage temperature and room temperature during aerobic exposure. When exposed to oxygen for 0 h, the temperature of the additive treatment was significantly higher than that of the control group, close to room temperature. At 36 h, the temperature of the CK group had increased significantly, and the temperature of each treatment started to be higher than room temperature. From 48 to 72 h, the temperature of the LP group continued to rise, while the other three groups tended to be stable. At 72 h, the temperature of each treatment group began to rise significantly. The temperature of the LP group first exceeded the room temperature by 2 °C at 84 h; the temperature of the CK group exceeded the room temperature by 2 °C at 120 h; the temperature of the PB group exceeded the room temperature by 2 °C at 180 h; the temperature of the LB group was 2 °C above room temperature. During the period from 48 h to 180 h, the temperature of the LP group was always higher than that of the other groups, and the temperature of the LB group was always lower than that of the other groups.

3.3. Changes in Antioxidant Capacity of Silage during Aerobic Exposure

Table 2. Effects of additives and days of aerobic exposure on antioxidant capacity of Leymus chinensis silage.

Items	Time (d)	Treatments (%)
		CK	LP	LB	PB
Total Phenol	0	0.1772 ± 0.0048 Da	0.3213 ± 0.0093 Ba	0.3358 ± 0.01 Aa	0.3037 ± 0.0048 Ca
	4	0.1015 ± 0.0065 Cb	0.1907 ± 0.0082 Bb	0.2176 ± 0.0065 Ab	0.2083 ± 0.0078 Ab
	8	0.0693 ± 0.0082 Cc	0.0953 ± 0.0078 Bc	0.1741 ± 0.0036 Ac	0.1668 ± 0.0048 Ac
Inhibition Rate	0	65.616 ± 0.0955 Ca	68.6406 ± 0.1988 Ba	71.0602 ± 0.2527 Aa	68.4814 ± 0.4963 Ba
	4	64.7883 ± 0.526 Ca	63.6103 ± 0.4963 Db	67.972 ± 0.4307 Ab	66.794 ± 0.2918 Bb
	8	63.5466 ± 0.526 Cb	62.8462 ± 0.5054 Cb	66.9214 ± 0.1459 Ac	65.425 ± 0.2527 Bc

Different capital letters indicate significant differences among different treatments under the same silage days (p < 0.05); different lowercase letters indicate significant differences among different silage days under the same treatments p < 0.05).

shows the total phenolic content and free radical inhibition rate of Leymus chinensis silage during aerobic exposure. At 0 days of aerobic exposure, the total phenolic content was significantly higher in the LB group and significantly lower in the CK group than in all other groups (p < 0.05). The total phenol content of all groups decreased significantly with the increase in aerobic exposure time (p < 0.05). At 8 days of aerobic exposure, the total phenol content of the LB and PB groups was significantly higher than that of the other groups (p < 0.05), and in the CK and LP groups, it decreased more. At 0 days of aerobic exposure, the inhibition rate of the LB group was significantly higher than that of the other groups, and that of the CK group was significantly lower than that of all other groups (p < 0.05). With the extension of aerobic exposure time, the inhibition rate of all groups decreased significantly (p < 0.05). During aerobic exposure, the inhibition rate of the LB group was always significantly higher than that of the other groups (p < 0.05), and the inhibition rate of the LP group decreased more.

3.4. Changes in Fatty Acids during Aerobic Exposure

The changes in fatty acids in Leymus chinensis silage during aerobic exposure are shown in

Table 3. Effects of additives and days of aerobic exposure on fatty acids of Leymus chinensis silage.

Items	Time (d)	Treatments (%)
		CK	LP	LB	PB
TFA	0	0.9705 ± 0.1504 Aa	1.0861 ± 0.1903 Aa	1.2443 ± 0.0871 Aa	1.0652 ± 0.1592 Aa
	4	0.9042 ± 0.0842 Aab	0.8636 ± 0.0821 Aab	1.1991 ± 0.2884 Aa	0.9391 ± 0.2053 Aa
	8	0.7358 ± 0.0831 Cb	0.8214 ± 0.0569 BCb	1.1802 ± 0.0328 Aa	0.8959 ± 0.1191 Ba
SFA	0	0.3917 ± 0.0805 Aa	0.4413 ± 0.1037 Aa	0.422 ± 0.0277 Aa	0.3962 ± 0.0264 Aa
	4	0.3783 ± 0.0374 Aa	0.356 ± 0.0263 Aa	0.4033 ± 0.0751 Aa	0.3689 ± 0.076 Aa
	8	0.2948 ± 0.0383 Ba	0.3686 ± 0.0115 Aa	0.3885 ± 0.0188 Aa	0.4212 ± 0.0589 Aa
PUFA	0	0.3967 ± 0.0516 Ba	0.3528 ± 0.0904 Ba	0.6464 ± 0.0884 Aa	0.5365 ± 0.1556 ABa
	4	0.3631 ± 0.0323 Ba	0.3551 ± 0.0184 Ba	0.5926 ± 0.1448 Aa	0.4118 ± 0.0667 Ba
	8	0.3558 ± 0.0448 Ba	0.3681 ± 0.0582 Ba	0.7014 ± 0.0165 Aa	0.374 ± 0.0364 B
MUFA	0	0.1821 ± 0.1201 Aa	0.2921 ± 0.1585 Aa	0.176 ± 0.0632 Aa	0.1324 ± 0.0204 Aa
	4	0.1627 ± 0.0452 Aa	0.1525 ± 0.0426 Aab	0.2032 ± 0.0808 Aa	0.1584 ± 0.0767 Aa
	8	0.0852 ± 0.0013 A	0.0847 ± 0.0116 Ab	0.0903 ± 0.0143 Aa	0.1007 ± 0.0246 Aa
C14:0	0	0.0415 ± 0.0045 Aa	0.0287 ± 0.0061 Bb	0.0371 ± 0.0019 Aa	0.0267 ± 0.0034 Bc
	4	0.0262 ± 0.0018 Bb	0.0354 ± 0.0019 ABab	0.0253 ± 0.0062 Bb	0.0399 ± 0.0085 Ab
	8	0.0277 ± 0.0022 Cb	0.0422 ± 0.0065 Ba	0.0425 ± 0.0023 Ba	0.0524 ± 0.0034 Aa
C15:0	0	0.002 ± 0.0004 Aa	0.0022 ± 0.0006 Aa	0.002 ± 0.0003 Aa	0.002 ± 0.0006 Aa
	4	0.0022 ± 0.0006 Aa	0.002 ± 0.0001 Aa	0.0022 ± 0.0004 Aa	0.0018 ± 0.0002 Aa
	8	0.0015 ± 0.0004 Aa	0.0018 ± 0 Aa	0.002 ± 0.0001 Aa	0.0022 ± 0.0006 Aa
C16:0	0	0.2778 ± 0.0448 Aa	0.3041 ± 0.0513 Aa	0.2976 ± 0.0137 Aa	0.2981 ± 0.0115 Aa
	4	0.2749 ± 0.0171 Aa	0.2544 ± 0.0114 Aa	0.2899 ± 0.0473 Aa	0.2548 ± 0.0468 Aa
	8	0.2232 ± 0.0327 Ba	0.2726 ± 0.0084 ABa	0.2898 ± 0.013 Aa	0.3079 ± 0.041 Aa
C16:1	0	0.025 ± 0.0081 Aa	0.0299 ± 0.0075 Aa	0.0271 ± 0.001 Aa	0.0292 ± 0.002 Aa
	4	0.0267 ± 0.0041 Aa	0.0223 ± 0.0022 Aa	0.0324 ± 0.0091 Aa	0.0225 ± 0.004 Aa
	8	0.0179 ± 0.0019 Ba	0.0214 ± 0.0008 ABa	0.0247 ± 0.0017 Aa	0.0251 ± 0.0044 Aa
C17:0	0	0.0043 ± 0.0022 Aa	0.0064 ± 0.0043 Aa	0.0046 ± 0.0012 Aab	0.0038 ± 0.0009 Aa
	4	0.0044 ± 0.0011 ABa	0.0035 ± 0.0007 Ba	0.0057 ± 0.0006 Aa	0.0037 ± 0.0011 Ba
	8	0.0018 ± 0.0016 Aa	0.003 ± 0.0002 Aa	0.0032 ± 0.0002 Ab	0.0036 ± 0.0009 Aa
C18:0	0	0.0551 ± 0.0302 Aa	0.0934 ± 0.0479 Aa	0.0684 ± 0.0156 Aa	0.0529 ± 0.0117 Aa
	4	0.0591 ± 0.0149 Aa	0.0496 ± 0.0166 Aa	0.0694 ± 0.0244 Aa	0.0563 ± 0.0206 Aa
	8	0.0311 ± 0.0022 Aa	0.0365 ± 0.0035 Aa	0.039 ± 0.0052 Aa	0.0413 ± 0.0125 Aa
C18:1n9t	0	0.0107 ± 0.0046 Aab	0.0114 ± 0.0065 Aa	0.0048 ± 0.0028 Aab	0.0072 ± 0.0038 Aa
	4	0.0128 ± 0.0028 Aa	0.0076 ± 0.0015 Aa	0.01 ± 0.0043 Aa	0.0072 ± 0.0032 Aa
	8	0.0055 ± 0.0014 Bb	0.0071 ± 0.0007 Ba	0.0024 ± 0.0005 Cb	0.0108 ± 0.0019 Aa
C18:1n9c	0	0.1464 ± 0.1074 Aa	0.2508 ± 0.1468 Aab	0.1441 ± 0.0595 Aa	0.096 ± 0.0192 Aa
	4	0.1232 ± 0.0389 Aa	0.1226 ± 0.0407 Aa	0.1607 ± 0.0723 Aa	0.1287 ± 0.07 Aa
	8	0.0618 ± 0.0034 Aa	0.0562 ± 0.0107 Ab	0.0631 ± 0.013 Aa	0.0649 ± 0.0185 Aa
C18:2n6c	0	0.1679 ± 0.0357 ABa	0.1395 ± 0.0549 Ba	0.2422 ± 0.0484 Aa	0.1706 ± 0.0451 ABa
	4	0.1245 ± 0.0068 Ba	0.1544 ± 0.02 ABa	0.1791 ± 0.031 Aa	0.171 ± 0.028 Aa
	8	0.1235 ± 0.0157 Ba	0.1267 ± 0.0102 Ba	0.2194 ± 0.0105 Aa	0.1322 ± 0.0086 Ba
C18:3n3	0	0.2258 ± 0.0222 Ba	0.211 ± 0.0361 Ba	0.4042 ± 0.0483 Aa	0.3645 ± 0.1117 Aa
	4	0.2354 ± 0.0252 Ba	0.199 ± 0.0134 Ba	0.412 ± 0.1139 Aa	0.2389 ± 0.0396 Ba
	8	0.2306 ± 0.0302 Ba	0.2386 ± 0.0483 Ba	0.4814 ± 0.0056 Aa	0.2392 ± 0.0301 Ba
C20:0	0	0.0102 ± 0.0017 ABa	0.0064 ± 0.0016 Cb	0.0113 ± 0.0026 Aa	0.0074 ± 0.0014 BCb
	4	0.0071 ± 0.0006 Ba	0.0103 ± 0.0004 Aa	0.0068 ± 0.0016 Bb	0.0107 ± 0.0006 Aa
	8	0.0078 ± 0.0019 Ba	0.0097 ± 0.0004 ABa	0.0098 ± 0.0006 ABab	0.0113 ± 0.0022 Aa
C20:3n6	0	0.0031 ± 0.0004 Aa	0.0023 ± 0.0006 ABa	0	0.0014 ± 0.0014 Ba
	4	0.0032 ± 0.0006 Aa	0.0017 ± 0.0015 Aa	0.0016 ± 0.0014 Aa	0.002 ± 0.0003 Aa
	8	0.0016 ± 0.0014 Aa	0.0028 ± 0.0001 Aa	0.0005 ± 0.0009 Aa	0.0027 ± 0.0023 Aa
C22:0	0	0	0	0	0.0036 ± 0.0062
	4	0.0039 ± 0.0067	0	0.0029 ± 0.0049	0
	8	0	0	0	0
C24:0	0	0.0008 ± 0.0014 ABa	0	0.0011 ± 0.001 ABa	0.0018 ± 0.0002 Aa
	4	0.0006 ± 0.001 Aa	0.0008 ± 0.0014 ABb	0.0011 ± 0.001 Aa	0.0016 ± 0.0014 Aa
	8	0.0017 ± 0.0003 Ba	0.0027 ± 0.0005 Aa	0.0023 ± 0.0004 ABa	0.0025 ± 0.0005 ABa

Different capital letters indicate significant differences among different treatments under the same silage days (p < 0.05); different lowercase letters indicate significant differences among different silage days under the same treatments p < 0.05).

at 0 and 4 days of aerobic exposure. The TFA, SFA, and MUFA contents of each group were not significantly different (p > 0.05). The PUFA contents of the LB group were significantly higher than those of the CK group (p < 0.05), and the LB group had the highest TFA and PUFA contents. At 8 days of aerobic exposure, the TFA and PUFA contents were significantly higher in the LB group (p < 0.05), the SFA contents were significantly lower in the CK group than in the other groups (p < 0.05), and there was no significant difference in the MUFA contents of the groups (p > 0.05). During aerobic exposure, the total fatty acid content of each treatment group showed a decreasing trend, while the TFA and PUFA contents of the LB group remained at a high level, and the MUFA content of the LP group decreased rapidly.

During aerobic exposure, saturated fatty acids tended to decrease except for C14:0, C20:0, and C24:0, and the differences between treatments were not significant (p > 0.05). At 4 and 8 days of aerobic exposure, the C14:0 content was significantly higher in the additive groups than in the CK group (p < 0.05), the C20:0 content was significantly higher in the PB and LP groups (p < 0.05), and the C24:0 content was significantly higher in the LP group than in the CK group (p < 0.05). During aerobic exposure, polyunsaturated fatty acids showed a decreasing trend, and the C18:2n6c and C18:3n3 contents were significantly higher in the LB group than in the other treatment groups (p < 0.05). During aerobic exposure, monounsaturated fatty acids showed a decreasing trend, and the differences between treatments were not significant at 0 and 4 days of aerobic exposure (p > 0.05). At 8 days of aerobic exposure, the C16:1 content of the CK group was significantly lower than that of the additive groups (p < 0.05), and the C18:1n9t of the LB group was significantly lower than that of the other treatment groups (p < 0.05).

3.5. Changes in Amino Acids during Aerobic Exposure

The changes in amino acids in Leymus chinensis silage during aerobic exposure are shown in

Table 4. Effects of additives and days of aerobic exposure on amino acids of Leymus chinensis silage.

Items	Time (d)	Treatments (%)
		CK	LP	LB	PB
TAA	0	11.7488 ± 0.1357 Ca	14.0573 ± 0.0045 Aa	13.3961 ± 0.0775 Ba	13.4761 ± 0.1161 Ba
	4	10.5513 ± 0.1429 Cb	12.6188 ± 0.3121 ABb	12.9406 ± 0.1365 Ab	12.5660 ± 0.0550 Bb
	8	9.8805 ± 0.1025 Dc	11.1348 ± 0.1034 Cc	12.0638 ± 0.0189 Ac	11.7564 ± 0.2185 Bc
EAA	0	5.6304 ± 0.0154 Ca	6.5886 ± 0.1274 Aa	6.39 ± 0.0178 Ba	6.4056 ± 0.0625 Ba
	4	4.9617 ± 0.0523 Cb	5.9481 ± 0.1222 Bb	6.1855 ± 0.1317 Ab	5.8945 ± 0.0836 Bb
	8	4.7032 ± 0.0427 Cc	5.2953 ± 0.0510 Bc	5.7079 ± 0.0665 Ac	5.6156 ± 0.1282 Ac
NEAA	0	6.1184 ± 0.1491 Ca	7.4687 ± 0.1320 Aa	7.0061 ± 0.0721 Ba	7.0705 ± 0.0845 Ba
	4	5.5896 ± 0.0909 Bb	6.6707 ± 0.1904 Ab	6.7552 ± 0.0495 Ab	6.6716 ± 0.1385 Ab
	8	5.1772 ± 0.0598 Dc	5.8395 ± 0.0536 Cc	6.3559 ± 0.0661 Ac	6.1408 ± 0.0942 Bc
Arg *	0	0.5111 ± 0.0103 Ba	0.6931 ± 0.0258 Aa	0.6659 ± 0.0243 Aa	0.7021 ± 0.0056 Aa
	4	0.4678 ± 0.0164 Cb	0.6065 ± 0.0062 Bb	0.6395 ± 0.0244 Aa	0.5972 ± 0.0149 Bb
	8	0.3899 ± 0.0073 Cc	0.5620 ± 0.0168 Bc	0.5916 ± 0.0065 Ab	0.5704 ± 0.0110 Bc
His *	0	0.3469 ± 0.0127 Ca	0.3761 ± 0.0167 Ba	0.3882 ± 0.0046 Ba	0.4186 ± 0.0109 Aa
	4	0.3114 ± 0.0100 Bb	0.3431 ± 0.0066 Ab	0.3493 ± 0.0041 Ab	0.3362 ± 0.0140 Ab
	8	0.3215 ± 0.0096 Bb	0.3230 ± 0.0060 ABb	0.3381 ± 0.0081 Ab	0.3338 ± 0.0071 ABb
Ile *	0	0.4947 ± 0.0161 Ca	0.5784 ± 0.0254 Aa	0.5358 ± 0.0206 Ba	0.5440 ± 0.0128 ABa
	4	0.4717 ± 0.0058 Bb	0.4956 ± 0.0084 ABb	0.5095 ± 0.0318 Aa	0.4893 ± 0.0114 ABb
	8	0.4513 ± 0.0091 Bb	0.4580 ± 0.0169 Bc	0.5133 ± 0.0166 Aa	0.5038 ± 0.0184 Ab
Leu *	0	1.0950 ± 0.0508 Ba	1.2528 ± 0.0149 Aa	1.2301 ± 0.0377 Aa	1.2530 ± 0.0460 Aa
	4	0.9379 ± 0.0221 Bb	1.1472 ± 0.0421 Ab	1.2167 ± 0.0477 Aa	1.1412 ± 0.0424 Ab
	8	0.9096 ± 0.0050 Cb	1.0653 ± 0.0470 Bc	1.1660 ± 0.0438 Aa	1.1922 ± 0.0312 Aab
Lys *	0	0.6657 ± 0.0195 Ba	0.7865 ± 0.0108 Aa	0.7665 ± 0.0342 Aa	0.6877 ± 0.0384 Ba
	4	0.6216 ± 0.0130 Cb	0.6890 ± 0.0277 Bb	0.7483 ± 0.0211 Aa	0.6849 ± 0.0069 Ba
	8	0.6106 ± 0.0214 Cb	0.6483 ± 0.0206 Bb	0.7180 ± 0.0106 Aa	0.6676 ± 0.0095 Ba
Met *	0	0.1138 ± 0.0029 Ca	0.1394 ± 0.0087 Aa	0.1270 ± 0.0056 Ba	0.1500 ± 0.0041 Aa
	4	0.1037 ± 0.0030 Ab	0.1233 ± 0.0060 Ab	0.1227 ± 0.0059 Aab	0.1213 ± 0.0267 Aab
	8	0.0921 ± 0.0005 Bc	0.1063 ± 0.0083 ABc	0.1132 ± 0.0067 Ab	0.1096 ± 0.0134 Ab
Phe *	0	0.7860 ± 0.0185 Ba	0.9297 ± 0.0296 Aa	0.8080 ± 0.0251 Ba	0.8350 ± 0.0305 Ba
	4	0.6555 ± 0.0103 Cb	0.7436 ± 0.0251 Bb	0.7666 ± 0.0245 ABab	0.7913 ± 0.0245 Aab
	8	0.5844 ± 0.0033 Cc	0.6846 ± 0.0251 Bc	0.7608 ± 0.0130 Ab	0.7239 ± 0.0483 ABb
Thr *	0	0.5222 ± 0.0114 Ba	0.6331 ± 0.0329 Aa	0.6669 ± 0.0213 Aa	0.6497 ± 0.0211 Aa
	4	0.4651 ± 0.0159 Bb	0.6264 ± 0.0226 Aa	0.6358 ± 0.0355 Aa	0.6117 ± 0.0154 Aab
	8	0.4341 ± 0.0192 Bb	0.5166 ± 0.0113 Ab	0.5308 ± 0.0120 Ab	0.5518 ± 0.0454 Ab
Val *	0	1.0950 ± 0.0422 Ba	1.1995 ± 0.0470 Aa	1.2016 ± 0.0360 Aa	1.1656 ± 0.0133 Aa
	4	0.9270 ± 0.0352 Cb	1.1733 ± 0.0532 ABa	1.1971 ± 0.0282 Aa	1.1213 ± 0.0179 Bb
	8	0.9097 ± 0.0328 Bb	0.9313 ± 0.0224 ABb	0.9762 ± 0.0337 Ab	0.9626 ± 0.0174 ABc
Ala	0	1.1794 ± 0.0104 Ca	1.2946 ± 0.0580 Aa	1.2611 ± 0.0297 ABa	1.2058 ± 0.0430 BCa
	4	0.9946 ± 0.0517 Bb	1.1938 ± 0.0146 Ab	1.2281 ± 0.0185 Aa	1.1952 ± 0.0423 Aa
	8	0.9667 ± 0.0387 Db	1.1441 ± 0.0331 Bb	1.2220 ± 0.0295 Aa	1.0394 ± 0.0386 Cb
Asp	0	1.7929 ± 0.0667 Ca	2.1696 ± 0.0199 Aa	2.0194 ± 0.0192 Ba	1.9809 ± 0.0777 Ba
	4	1.7277 ± 0.0778 Ca	1.9231 ± 0.0265 Bb	2.0379 ± 0.0392 Aa	1.7277 ± 0.0778 ABa
	8	1.6787 ± 0.0651 Ba	1.7047 ± 0.0550 Bc	1.8970 ± 0.0903 Ab	1.8695 ± 0.0287 Ab
Cys	0	0.0767 ± 0.0022 Ba	0.0862 ± 0.0020 Aa	0.0779 ± 0.0022 Ba	0.0789 ± 0.0011 Ba
	4	0.0627 ± 0.0016 Bb	0.0685 ± 0.0033 Ab	0.0698 ± 0.0019 Ab	0.0686 ± 0.0034 Aa
	8	0.0599 ± 0.0007 Bb	0.0604 ± 0.0013 Bc	0.0678 ± 0.0009 Ab	0.0645 ± 0.0032 Ab
Glu	0	1.1907 ± 0.0664 Ca	1.5667 ± 0.0489 Aa	1.3721 ± 0.0291 Ba	1.5129 ± 0.0491 Aa
	4	1.0967 ± 0.0541 Ca	1.2873 ± 0.0183 ABb	1.2181 ± 0.0386 Bb	1.3052 ± 0.0307 Ab
	8	0.8386 ± 0.0172 Cb	1.0834 ± 0.0206 Bc	1.1535 ± 0.0518 Ab	1.1410 ± 0.0144 Ac
Gly	0	0.6498 ± 0.0320 Ba	0.6817 ± 0.0261 Aa	0.6901 ± 0.0116 Ba	0.6827 ± 0.0526 Ba
	4	0.6295 ± 0.0187 Ba	0.6077 ± 0.0073 Aab	0.6273 ± 0.0054 Ab	0.6285 ± 0.0063 Ab
	8	0.6194 ± 0.0073 Ba	0.6055 ± 0.0223 Bb	0.6226 ± 0.0053 Ab	0.6132 ± 0.0155 Ac
Pro	0	0.5752 ± 0.0135 Ca	0.7121 ± 0.0145 Aa	0.7033 ± 0.0224 ABa	0.6749 ± 0.0167 Ba
	4	0.4914 ± 0.0109 Bb	0.6553 ± 0.0277 Ab	0.6811 ± 0.0228 Ab	0.6588 ± 0.0210 Aa
	8	0.4792 ± 0.0319 Bb	0.5247 ± 0.0354 Bc	0.6177 ± 0.0191 Ab	0.5899 ± 0.0308 Ab
Ser	0	0.2796 ± 0.0073 Ca	0.4831 ± 0.0169 Aa	0.4238 ± 0.0209 Ba	0.4588 ± 0.0131 Aa
	4	0.2470 ± 0.0082 Cb	0.3815 ± 0.0275 Bb	0.4187 ± 0.0156 Aa	0.4263 ± 0.0057 Ab
	8	0.2343 ± 0.0090 Cb	0.3596 ± 0.0059 Bb	0.3931 ± 0.0105 Aa	0.3739 ± 0.0194 ABc
Tyr	0	0.3742 ± 0.0046 Ba	0.4413 ± 0.0102 Aa	0.3909 ± 0.0087 Ba	0.4254 ± 0.0116 Aa
	4	0.3399 ± 0.0125 Bb	0.3769 ± 0.0089 Ab	0.3742 ± 0.0139 Aab	0.3798 ± 0.0179 Ab
	8	0.3004 ± 0.0127 Cc	0.3238 ± 0.0094 Bc	0.3654 ± 0.0091 Ab	0.3488 ± 0.0033 Ac

Different capital letters indicate significant differences among different treatments under the same silage days (p < 0.05); different lowercase letters indicate significant differences among different silage days under the same treatment p < 0.05).

. At 0 days of aerobic exposure, the TAA, EAA, and NEAA contents were significantly higher in the LP group and significantly lower in the CK group than in the other groups (p < 0.05). With the prolongation of aerobic exposure, the TAA, EAA, and NEAA contents in all groups decreased significantly. At 4 days of aerobic exposure, the TAA, EAA, and NEAA contents in the LB group were significantly higher than those in the CK group (p < 0.05). At 8 days of aerobic exposure, the LB group had contents significantly higher than those in the CK and LP groups (p < 0.05), and the decrease in amino acid contents in the LP group was greater.

At 0 days of aerobic exposure, the contents of various amino acids in the additive group were significantly higher than those in the CK group (p < 0.05), except for Lys, Phe, Cys, Gly, and Tyr. Among them, Phe, Asp, Cys, Gly, Ser, and Tyr contents were significantly higher in the LP group (p < 0.05), Ile, Met and Glu contents were significantly higher in LP and PB groups (p < 0.05), Lys contents were significantly higher in the LP and LB groups, and His contents were significantly higher in PB group (p < 0.05). With the prolongation of aerobic exposure time, the amino acid contents of all groups decreased to different degrees. At 8 days of aerobic exposure, the contents of Arg, Lys, and Ala in the LB group were significantly higher than those in the other groups (p < 0.05), the contents of Ile, Leu, Asp, Glu, Gly, Pro, and Tyr in the LB and PB groups were significantly higher than those in the other groups (p < 0.05), and the amino acid contents in the CK and LP groups decreased to a greater extent.

3.6. Correlation of Microbial Genera Levels with Fatty Acids and Amino Acids during Aerobic Exposure

Figure 2A shows the correlation between bacterial genus levels and fatty acids during aerobic exposure. The Lactobacillus was significantly positively correlated with C16:0 (p < 0.05). Sphingomonas was significantly positively correlated with C24:0 and significantly negatively correlated with MUFA (p < 0.05). Prevotella was significantly negatively correlated with C18:3n3 and PUFA (p < 0.05). Weissella was significantly negatively correlated with C16:1 and TFA (p < 0.05). Lactococcus, Brevundimonas, Clostridium, Devosia, and Enterococcus were significantly negatively correlated with C16:0 (p < 0.05). Brevundimonas, Clostridium, Klebsiella, Pantoea, Devosia, and Enterococcus were significantly positively correlated with C15:0 (p < 0.05).

Figure 2B shows the correlations between levels of fungal genus and fatty acids during aerobic exposure. Cryptococcus was significantly positively correlated with TFA, UFA, MUFA, C18:1n9c, and C17:0 (p < 0.05). Thermoascus was significantly positively correlated with MUFA, C18:1n9c, and C18:0 (p < 0.05). Penicillium and Monascus were significantly positively correlated with C14:0 and C24:0 and negatively correlated with MUFA, C18:0, and C18:1n9c (p < 0.05). Debaryomyces, Glomerella and Sarociadium were positively correlated with PUFA, C18:3n3c, and C18:2n3c (p < 0.05).

Figure 3A shows the correlations between bacterial genus levels and amino acids during aerobic exposure. Lactobacillus was extremely significantly positively correlated with TAA, EAA, and NEAA (p < 0.01), and Enterococcus was extremely significantly negatively correlated with TAA, EAA, and NEAA (p < 0.01). Lactococcus and Levilactobacillus were extremely significantly negatively correlated with TAA, EAA, and NEAA (p < 0.01). Actinomyces was significantly negatively correlated with EAA (p < 0.05) and was extremely significantly negatively correlated with TAA and NEAA (p < 0.01). Lactobacillus was significantly positively correlated with Lys, Gly, His, Val, and Ala (p < 0.05) and was extremely significantly positively correlated with other amino acids (p < 0.01). Neisseria was significantly positively correlated with Glu and Ser (p < 0.05). Lactococcus was significantly negatively correlated with all amino acids except Gly, His, Cye, and Ile (p < 0.05). Actinomyces was significantly negatively correlated with all amino acids except Lys, Gly, His, Cye, and Ile (p < 0.05). Levilactobacillus was significantly negatively correlated with all amino acids except Lys, Gly, Asp, and Val (p < 0.05). Brevundimonas, Devosia, Enterococcus and Clostridium were significantly negatively correlated with Phe, Glu, and Arg (p < 0.05). Klebsiella and Enterobacter were significantly negatively correlated with Thr, Glu, and Arg (p < 0.05).

Figure 3B shows the correlations between fungal genus levels and amino acids during aerobic exposure. Penicillium was significantly negatively correlated with NEAA (p < 0.05). Simplicillium was significantly positively correlated with TAA, EAA, and NEAA (p < 0.05), and Malassezia was significantly positively correlated with TAA and EAA (p < 0.05). Penicillium was significantly negatively correlated with Val, Asp, Pro, Ala, Met, Cys, Phe, and Tyr (p < 0.05). Simplicillium was significantly positively correlated with Thr, Val, Asp, Pro, Lys, His, Glu, Ile, and Cys (p < 0.05). Malassezia was significantly positively correlated with Thr, Val, and Ala (p < 0.05). Trichosporon and Thermoascus were significantly positively correlated with Ile, Cys, Phe, and Tyr (p < 0.05). Cochliobolus and Villosiclava were extremely significantly positively correlated with Gly, His, and Met (p < 0.01).

4. Discussion

Analysis of the fatty acids, amino acids, and antioxidant capacity of the silage feedstock showed that the total fatty acid content of the Leymus chinensis feedstock was low but the proportion of unsaturated fatty acids was high. All of the Leymus chinensis total fatty acids decreased to different extents following silage fermentation, but there was an increase in the proportion of saturated fatty acids which could be caused by the oxidation of unsaturated fatty acids by microorganism-produced lipase, which is in agreement with the findings of Liu et al. [22]. The amino acid content of the Leymus chinensis raw material was higher and the proportion of non-essential amino acids was higher. The amino acid content of Leymus chinensis increased after silage fermentation as some proteins were broken down into amino acids by the action of plant cell enzymes, while some microorganisms required amino acids as nitrogen sources for their synthesis of bacteriophage proteins, leading to changes in amino acid types and proportions that were in agreement with the findings of Guo et al. [23] and Muck et al. [24]. After ensiling, Leymus chinensis showed different degrees of decrease in total phenolic content and free radical inhibition, which could be due to oxidative decomposition early in fermentation and metabolic conversion of microbes during fermentation, which is in agreement with the findings of He et al. [25].

Interestingly, the temperature of the LP group was the first to rise 2 °C above room temperature during the aerobic exposure period, and the temperature of the LB group was the last to exceed ambient temperature by 2 °C. This suggests that Lentilactobacillus buchneri can indeed inhibit silage fever, which is in agreement with the results of Gallo et al. [26]. The LP group showed the fastest temperature increase, which may be caused by heat generated during the growth and reproduction of yeast utilizing lactic acid and miscellaneous bacteria utilizing soluble sugars, which is in agreement with the findings of Wang et al. [27].

The total phenolic content and free radical inhibition rate during aerobic exposure showed a decreasing trend in all groups, but those of the LB group were significantly higher than the other groups, and they were significantly lower in the CK group than in other groups. This was due to oxygen seepage after the silage was opened activating aerobic microorganism reproduction, and the acetic acid produced by Lentilactobacillus buchneri was found to be bacteriostatic, which could effectively inhibit the growth and metabolism of various bacteria as well as reduce the loss and denaturation of antioxidant substances [28]. In the case of the LP group, the greater degree of decrease in total phenolic content and inhibition following aerobic exposure was because phenols are more easily oxidized at higher pH when exposed to air [29], and the lactic acid produced by Lactiplantibacillus plantarum becomes a substrate for growth of the aerobic bacteria in the aerobic phase, accelerating their oxidative nutrient decomposition, which is in agreement with the conclusions of Mugabe et al. [30].

Analysis of the fatty acid changes in each treatment for aerobic exposure demonstrated that the total fatty acid content of the additive group was greater than that of the CK group at 0 days of aerobic exposure, and total and unsaturated fatty acids in the LB group were highest, which may be caused by the inhibitory effect of Lentilactobacillus buchneri on microorganisms that utilize fatty acids [22]. Considering the unsaturated fatty acids, the LB group had the highest content of polyunsaturated fatty acids, and the LP group had the highest value of monounsaturated fatty acids, which may be a result of the high fermentation of Lactiplantibacillus plantarum that results in more monounsaturated fatty acids, which is in agreement with the findings of Zhang et al. [31]. Total fatty acid content within each treatment group showed a decreasing trend at 4 and 8 days of aerobic exposure, and the total fatty acid content in the LB group remained at a high level, while the monounsaturated fatty acid in the LP group decreased rapidly. This is because monounsaturated fatty acids themselves are automatically oxidized, and when the heat reaches a certain limit, oxidation is accelerated, resulting in the breaking of unsaturated double bonds and the generation of harmful substances such as aldehydes and ketones [32]. The aerobic stability of the LP group was poor, and the temperature rose faster, promoting the growth of aerobic bacteria and the oxidation process of monounsaturated fatty acids [33].

C18:1n9c content was greater in the LP group at 0 days of aerobic exposure, and the C18:2n6c and C18:3n3 contents in the LB group were greater; C18:1n9c content decreased in the LP group at 4 and 8 days of exposure to aerobic exercise, and the C18:2n6c and C18:3n3 contents in the LB group remained elevated. Of these, C18:2n6c and C18:3n3 are beneficial for ruminants and have the potential to reduce arteriosclerosis in animals [34]. Lentilactobacillus buchneri alone was more effective in retaining unsaturated fatty acids, and their content in the other treatment groups decreased severely to levels even lower than in the fresh samples, which is in agreement with the findings of Alves et al. [35]. The content of C18:1n9c can be increased after ensiling compared with fresh samples, but it is readily oxidized during aerobic exposure, resulting in a rapid decrease in content [36].

Analysis of the amino acid changes in each treatment for aerobic exposure showed that the LP group had the highest total amino acid content at 0 days of aerobic exposure and that the LB group had the highest total amino acid content at 8 days of aerobic exposure. This is because proteolysis and degradation of amino acids occur in the process of ensiling under the action of microorganisms, and the lactic acid produced by Lactiplantibacillus plantarum can accelerate the fermentation process and reduce the loss of amino acids by miscellaneous bacteria in the early stage of fermentation [37]. As bacterial growth and metabolism produce large amounts of proteases, they hydrolyze the proteins in the feed into amino acids, which are denatured by oxidative reactions under aerobic exposure, producing urea, water, carbon dioxide, and heat [38]. However, Lentilactobacillus buchneri can enhance the aerobic stability of silage, thereby reducing the breakdown and utilization of proteins and amino acids by aerobic bacteria during aerobic exposure, but Lactiplantibacillus plantarum is less effective at improving the aerobic stability of silage, leading to greater nutritional losses [18,39].

At 0 d of aerobic exposure, the amino acid abundances of Phe, Asp, Cys, Gly, Ser, Tyr, Ile, Met, Glu, Lys, and His were increased to different extents in the additive group, indicating that amino acid content could be effectively increased through the use of additives to lactic acid bacteria, which is in agreement with the results of Lim et al. [40]. These include phenylalanine and tyrosine, which synthesize important neurotransmitters and hormones in cattle and are involved in the metabolism of glucose and adipose tissue [41]. Aspartic acid contributes to immunoglobulin and antibody production to enhance immunity in livestock [42]. The combination of glycine and serine can reduce the concentration of cholesterol in the blood of cattle and prevent and treat high blood pressure [43]. Histidine can be added to the diet to improve animal performance, increase muscle sarcopeptide content, and increase the body’s antioxidant capacity [44]. The concentrations of Arg, Lys, and Ala were higher in the LB group after 8 d of aerobic exposure. Arginine has been shown to have an antioxidant effect and can reduce the oxidation of low-density lipoproteins [45]. In animals, Lysine is one of the essential amino acids and it can enhance growth and development, increase immune function, and enhance central nervous tissue function [46]. The LB group and PB group have higher Ile and Leu contents. Isoleucine, valine, and leucine, collectively referred to as branched-chain amino acids, make important contributions to growth, development, and reproduction, promote muscle growth and recovery, and prevent muscle breakdown and oxidation for energy supply [47].

Analysis of the correlation between bacterial genus levels and fatty acids during aerobic exposure shows that Lactobacillus positively correlated with most of the fatty acids, which is because Lactiplantibacillus plantarum in Lactobacillus can rapidly reduce pH and inhibit the activity of oxidase in silage. The acetic acid produced by Lentilactobacillus buchneri during aerobic exposure might inhibit the growth of various bacteria and ensure the type and content of the fatty acids, which is in agreement with research findings by Ke et al. [48]. Weissella is negatively correlated with both total fatty acids and unsaturated fatty acids, and the research of Zong et al. [49] shows that Weissella reduces the activity of lipase, thereby inhibiting its decomposition of fat and reducing the production of polyunsaturated fatty acids.

The correlation analysis between fungal genera level and fatty acids during aerobic exposure showed that Cryptococcus, Penicillium, and Monascus were closely related to fatty acid changes. Cryptococcus includes many lipid yeasts, which can produce saturated and monounsaturated long-chain fatty acids, especially oleic acid [50]. Thermoascus can use part of the unsaturated fats and convert them into saturated fatty acids, especially palmitate, oleic acid and stearic acid [51]. The metabolism of Penicillium and Monascus is related to the breakdown of protein, fat, and sugar. With the prolongation of fermentation time, the content of total free fatty acids and unsaturated fatty acids increased significantly, which is consistent with the research results of Xia et al. [52]. Li et al. [53] showed that Debaryomyces and Wesseria are associated with free amino acids and free fatty acids and produce volatile fatty acids that affect flavor. These bacterial and fungal genera may have a strong influence on the type and content of fatty acids in silage and during aerobic exposure of Leymus chinensis.

The correlation analysis between the levels of bacterial genera and amino acids during aerobic exposure showed that Lactobacillus, Enterococcus, Lactococcus, and Levilactobacillus correlated more strongly with TAA, EAA, and NEAA, probably due to the rapid decrease in the number of Lactobacillus after opening and the competitive relationships it has with Enterococcus, Lactococcus, and Levilactobacillus The relative increase in numbers is consistent with the findings of Zhao et al. [54]. The growth and metabolism of Actinobacillus, a common aerobic bacterium in the silage process, causes the depletion of protein, amino acids, and soluble carbohydrates from the silage [55]. Clostridium can break down sugars, organic acids and proteins, producing unpleasant butyric acid and ammonia, among others. The main harmful effect of Clostridium is to cause protein fermentation and decomposition, and the main fermentation pathways that have been clarified are firstly, releasing ammonia from amino acids, leaving organic acid residues, and secondly, using amino acids to form amines or oxidizing and reducing amino acids to fatty acids, thereby significantly affecting the type and content of amino acids [56,57]. Enterobacter and Klebsiella are often found in the pre-silage and higher pH stages after aerobic exposure and are mainly associated with diamino and decarboxylated amino acids in the silage, which can reduce the aerobic stability of the silage [58].

The correlation analysis between the level of fungal genera and fatty acids during aerobic exposure showed that Penicillium is one of the main fungal genera causing silage deterioration by breaking down proteins into peptides, amino acids, amines, etc. At the same time, some strains produce toxins that affect the central nervous system and seriously endanger the health of livestock [59]. Most groups of Malassezia produce lipase, which can break down lipids into fatty acids and provide nutrition for their metabolism [60]. The warm, humid, and aerobic environment after silage opening provides conditions for Thermoascus and Trichosporon to develop, producing enzymes that promote proteolysis and amino acid conversion [61,62]. Cochliobolus and Villosiclava are common pathogens in the field and often cause spoilage diseases in crops and can also cause spoilage and nutrient breakdown in silage [63]. These bacterial and fungal genera may have a strong influence on the type and content of amino acids in silage and during aerobic exposure of Leymus chinensis.

5. Conclusions

This study showed that fatty acid, amino acid, total phenolic content and free radical inhibition decreased to different degrees in all groups during aerobic exposure of Leymus chinensis silage, with smaller decreases in the LB group. At 8 days of aerobic exposure, the fatty acid, amino acid, total phenol content, and the rate of inhibition of free radicals decreased to different extents in all groups, with the LB group exhibiting a lesser decline. Correlation analysis showed that Lactobacillus, Weissella, Cryptococcus, Penicillium, and Rhodococcus were more correlated with fatty acid changes, and Lactobacillus, Actinobacillus, Clostridium, Penicillium, and Thermoascus were more correlated with amino acid changes. In conclusion, the addition of Lentilactobacillus buchneri could effectively improve the antioxidant capacity, and fatty acid and amino acid contents of Leymus chinensis silage and during aerobic exposure, while Lactiplantibacillus plantarum alone was able to effectively improve upon the above indices following ensiling, but the degree of decline was greater during aerobic exposure.