Vitamin A in total mixed ration silage influenced by various lactic acid bacteria: Lactobacillus plantarum, Lactobacillus casei and Enterococcus faecium

doi:10.21203/rs.3.rs-4972300/v1

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Vitamin A in total mixed ration silage influenced by various lactic acid bacteria: Lactobacillus plantarum, Lactobacillus casei and Enterococcus faecium

https://doi.org/10.21203/rs.3.rs-4972300/v1

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This paper focuses on the impact of various strains of lactic acid bacteria (LAB) on vitamin A content in total mixed ration (TMR) silage. TMRs were respectively inoculated with Lactobacillus plantarum (LP), Lactobacillus casei (LC), and Enterococcus faecium (EF) and then ensiled for 56 days in the dark and anaerobic conditions. During ensiling, the fermentation quality, chemical composition, and microbial composition of TMR silage were analyzed, as well as the vitamin A. In addition, the free radical scavenging ability, total antioxidant capacity (T-AOC) and antioxidant enzyme activities (SOD, GSH-Px, CAT) of the three LAB strains were investigated. After 56 days of ensiling, the activities of T-AOC, SOD, GSH Px and CAT of TMR silage were also analysed. The results showed that LAB had significant effects on the fermentation quality and vitamin A of TMR silage (p<0.05). LP and LC could enhance the fermentation quality of TMR. Vitamin A loss rates in decreasing order were LC, CK, EF and LP groups. LAB endowed with elevated antioxidant properties are capable of augmenting the antioxidant capacity of total mixed ration (TMR) silage, thereby mitigating the degradation of vitamin A.

Biological sciences/Microbiology

Earth and environmental sciences/Environmental sciences

LAB

Vitamin A

TMR silage

Antioxidant activity

In recent decades, the livestock industry has undergone significant transformations in response to evolving consumer demands, environmental concerns, and scientific advancements. Among the many facets of livestock management, nutrition stands out as a critical determinant of animal health, productivity, and the quality of animal-derived products. In the field of nutrition, the potential of silage, particularly total mixed ration (TMR) silage, to optimise feed efficiency, enhance nutrient utilisation and mitigate feed spoilage has attracted considerable attention [1–2]. TMR silage is a fermented feed that is commonly used in animal husbandry, particularly for the feeding of ruminants. It is scientifically formulated with multiple ingredients based on the animal's nutritional needs, including roughage, concentrates, minerals, vitamins and other additives [3]. Therefore, TMR silage can ensure that every bite of feed consumed by animal is a complete diet, with a stable ratio of concentrate to roughage and a consistent nutrient concentration. In addition, TMR silage is fermented by lactic acid bacteria (LAB) under anaerobic condition, which play a pivotal role in converting sugars into organic acids, primarily lactic acid. This lowers the pH of the TMR silage, thereby inhibiting the growth of undesirable microorganisms and preserving nutrient integrity [4]. However, beyond its role in ensiling, recent research has begun to shed light on the impact of LAB on the nutritional composition of TMR silage, with particular emphasis on essential micronutrient such as vitamin A. Vitamin A, a fat-soluble micronutrient, plays a fundamental role in various physiological processes within the animal body, including vision, immune function, and reproduction [5]. However, ruminant animals are unable to synthesize vitamin A and must therefore obtain it from their feed. Consequently, it is essential to prevent the loss of vitamin A in TMR silage to ensure that the animals are getting an adequate amount of vitamin A.

In previous study, Tian et al. (2023) found LAB are associated with the loss of vitamin A throughout the fermentation process of TMR [6]. The acidic environment, which results from the metabolic activities of LAB, has been indicated to diminish the vitamin A content in TMR silage [7]. However, the fermentation of TMR is a biochemical process. It is unknown whether there are other factors involved in losing vitamin A caused by LAB. Thus, the objective of this paper is to investigated influence of various LAB on vitamin A in TMR silage.

The effectiveness of LAB as additives in TMR silage applications is widely recognized [8]. Lactobacillus plantarum (LP) is frequently incorporated into TMR silage to enhance fermentation quality, due to its rapid growth and capacity to swiftly reduce pH levels [9–10]. Lactobacillus casei (LC) demonstrates excellent acid resistance and is capable of significantly reducing the ammonia nitrogen (NH₃-N) content in silage [11]. Enterococcus faecium (EF) has a good ability to degrade carbohydrates, which can improve the utilization efficiency of carbohydrates in ruminants [12]. Therefore, the various LAB exhibit disparate functions. This study posited the hypothesis that distinct species of LAB exert variable influences on the vitamin A content in TMR silage, with the aim of elucidating the underlying mechanisms through which LAB impacts vitamin A preservation.

2.1 LAB activated

The strains LP, LC, and EF, derived from TMR silage, were cryopreserved at -80°C within a cryoprotectant solution containing 10% glycerol. Subsequently, these LAB were individually inoculated into 5 milliliters of de Man, Rogosa, and Sharpe (MRS) broth. This culture was maintained at 37°C for 24 hours. Following this initial incubation, the cultures underwent two subsequent passages. They were then transferred into 50 milliliters of fresh MRS broth and incubated under the same conditions for another 24-hour period. The population of LAB was quantified employing the serial dilution technique, which facilitated the calculation of the appropriate dosage for subsequent applications.

2.2 TMR and TMR silage preparation

TMR was prepared by combining alfalfa hay, soy milk residue, soybean meal, maize flour, vitamin-mineral supplements, and salt in the ratio of 36:21:5:32.5:5:0.5 based on dry matter (DM) content. Following the mixture, the strains LP, LC, and EF were introduced into the TMR at a concentration of 10⁶ colony-forming units per gram of fresh weight (cfu/g FW). A control group (CK) received an equivalent volume of sterile water. Each treatment was replicated three times, with 300 grams of the mixture placed into vacuum-sealable bags. The fermentation process for all treatments was conducted at ambient temperature, ranging from 24 to 28°C, in a dark environment.

2.3 Chemical Analysis

The DM content was ascertained by drying the sample at 65°C for a duration of 48 hours, in accordance with the protocol outlined in the 930.15 method [13]. Post-drying, the samples underwent mechanical pulverization to achieve a particle size that could pass through a 1-mm mesh, utilizing a laboratory-grade knife mill. The resultant fine powder was subsequently employed for the quantification of water-soluble carbohydrates (WSC), crude protein (CP), neutral detergent fiber (NDF), and acid detergent fiber (ADF). The WSC was evaluated employing the anthrone assay as described by [14]. The CP was quantified adhering to the analytical procedures set forth by the Association of Official Analytical Communities (AOAC), as detailed in Yang et al. (2019) [15]. The determination of NDF and ADF was conducted following the technique delineated by Van Soest, Robertson and Lewis (1991) [16]. Vitamin A is tested using fresh samples after freeze-drying using a vacuum freeze-dryer, with reference to the testing method of Tian et al. (2020) [7].

2.4 Microbial Analysis

Serial dilutions ranging from 10^− 1 to 10^− 7 were crafted from the water extract to enumerate LAB, aerobic bacteria, and yeasts. The enumeration of LAB was executed on MRS agar medium (Difco Laboratories, Detroit, MI, USA) and proceeded with incubation under anaerobic conditions at 37°C for a period of 48 hours. For aerobic bacteria, a nutrient agar medium (Nissui, Tokyo, Japan) was utilized, with incubation at 30°C for 24 hours prior to counting. Yeast enumeration was carried out on potato dextrose agar (Nissui, Tokyo, Japan) followed by incubation at 30°C for 48 hours before the quantification of colonies.

2.5 LAB antioxidant activity determination

2.5.1 Preparation of cells and intracellular cell-free extracts

LAB were diluted to a 10-fold concentration with phosphate-buffered saline (PBS) solution, homogenized for a minute using a turbine mixer, and subsequently inoculated into MRS broth at a 2% volume-to-volume ratio under conditions devoid of oxygen and maintained at 37°C for a duration of 12 hours. The bacterial cells were then collected by centrifugation at a force of 6000g for a period of 10 minutes at 4°C, followed by washing twice with deionized water and resuspension in the same. The concentration of the bacterial suspension was standardized to 10⁹ cfu/mL.

To prepare intracellular cell-free extracts, a procedure was adapted with minor alterations from Lin and Yen (1999) [17]. The cells were treated with lysozyme (1 mg/mL) and kept at 37°C for 30 minutes. Subsequently, ultrasonic disruption was applied using a sonicator. The sonication process involved five cycles of 1-minute duration, with intervals in an ice bath to prevent overheating. The cell debris was eliminated through centrifugation at 8000g for 10 minutes at a temperature of 4°C, yielding the supernatant as the intracellular cell-free extract.

2.5.2 Scavenging of DPPH free radical

The ability of LAB to neutralize DPPH radicals was assessed by employing a refined adaptation of the technique originally described by Kao and Chen (2006) [18]. Initially, 1.0 mL of a LAB suspension at a concentration of 10⁹ cfu/mL was combined with 2.0 mL of an ethanolic solution of DPPH radicals at a concentration of 0.05 mmol/L. The resulting mixture was homogeneously mixed and then kept under ambient temperature within a darkened setting for a duration of 30 minutes. Control samples were prepared with deionized water and DPPH solution, while blank samples contained ethanol and cells without DPPH. Post incubation, the mixture was underwent centrifugation at 8000g for 10 minutes to separate the components. The absorbance of the supernatant was conducted in triplicate at a wavelength of 517 nm employing a spectrophotometer.

The calculation of the scavenging capacity was executed utilizing the formula:

Scavenging activity (%)=[1- (A_s-A_b)/A_c] × 100

In this equation, A_s, A_b, and A_c denote the absorbance values of the sample, the blank, and the control, respectively.

2.5.3 Scavenging of hydroxyl radical (•OH)

The assay for •OH scavenging was executed utilizing the Fenton reaction approach. Succinctly, the reaction solution was composed of 1.0 mL of a brilliant green solution at a concentration of 0.435 mmol/L, 2.0 mL of ferrous sulfate (FeSO₄) at 0.5 mmol/L, 1.5 mL of hydrogen peroxide (H₂O₂) at a 3.0% mass/volume ratio, and 1.0 mL of the intracellular cell-free extract. This mixture was kept at ambient temperature for a period of 20 minutes, after which the absorbance was recorded at a wavelength of 624 nm. The degree of absorbance alteration in the reaction mixture served as an indicator of the LAB strains' capacity to neutralize •OH. The efficacy of •OH scavenging was determined by applying the following formula:

Scavenging activity (%) = [(As-A_C)/(A -A_C)] ×100

The absorbance measurements are defined by the following: A_S corresponds to the absorbance recorded in the presence of the sample; A_C is the absorbance of the control group, which lacks the sample; and A represents the absorbance measured in the absence of both the sample and the Fenton reaction system.

2.5.4 Superoxide anion (O²⁻) scavenging ability

A mixture comprising Tris-HCl buffer (150 mmol/L, pH 8.2), EDTA (3 mmol/L), 1,2,3-benzenetriol (1.2 mmol/L), and cell-free extract (0.5 mL) was prepared, achieving a cumulative reaction volume of 3.5 mL, as per the method described by Oyaizu (1986) [19]. This concoction was then subjected to incubation at 25°C for 10 minutes. Subsequent to the incubation period, the absorbance of the mixture was determined at a wavelength of 325 nm applying a spectrophotometer. The capability of the cell-free extract to counteract O²⁻was quantified by calculating the percentage of inhibition as follows:

Scavenging effect (%) =[1- (A_D -A_B)/(A_A- A_C)] ×100

The absorbance values are designated as follows: A_A represents the absorbance of a sample devoid of both cell-free extract and 1,2,3-benzenetriol; A_C signifies the absorbance of a sample without cell-free extract but in the presence of 1,2,3-benzenetriol; A_B denotes the absorbance of a sample containing cell-free extract yet absent of 1,2,3-benzenetriol; and A_D is indicative of the absorbance of a sample containing both cell-free extract and 1,2,3-benzenetriol.

2.5.5 Tolerance to hydrogen peroxide

LAB were inoculated in the medium containing H₂O₂ (2 mmol/L) and incubated at 37°C for 24 h. The cell concentration (expressed as OD600 value) was determined (van Niel, Hofvendahl and Hahn-Hägerdal 2002)[20].

2.5.6 Determination of antioxidant activity of LAB

The total antioxidant capacity (T-AOC) and three antioxidant enzymes of three LAB and the activity of, including superoxide dismutase (SOD) activity, catalase (CAT), and glutathione peroxidase (GSH Px), were analyzed using a reagent kit according to the instructions.

2.5.7 Determination of antioxidant activity of TMR fermentation

Following the procedure described by Zhang et al. (2020), the TMR silage samples were processed by centrifugation at a force of 12000g for a duration of 10 minutes at a temperature of 4°C [21]. The supernatant obtained from this process was subsequently utilized for the assessment of T-AOC, SOD activity, GSH Px activity, and CAT activity.

2.6 Statistical Analysis

Each test was performed three times to ensure accuracy and reliability of the results. Statistical comparisons were made using SPSS20.0 software.

To ascertain the statistical significance of the disparities among the various treatments, an analysis of variance (ANOVA) was employed. Subsequently,

Tukey's multiple comparison method was utilized to delineate the specific contrasts. The threshold for statistical significance was established at p < 0.05. The correlation between vitamin A and the array of measured variables was evaluated using the Pearson correlation coefficient. The criterion for statistical significance was set at p < 0.05.

3.1 Fermentation quality and microbial composition of TMR

The variations in fermentation quality and the microbial profile throughout the ensiling process of TMR supplemented with distinct LAB were delineated in Tables 1 and 2, respectively. The type of LAB, ensiling time, and their interaction have significant effects on pH, acetic acid, NH₃-N, and LAB number (p < 0.05). Lactic acid is affected by the type of LAB and ensiling time (p < 0.05). Along with the fermentation, the pH of all TMRs decreased significantly, especially in the LP and LC groups. However, the content of lactic acid and acetic acid increased, and butyric acid was never detected. The NH₃-N content of all TMRs gradually increased during the fermentation, but was lower than 5% TN at 56 days of fermentation. The NH₃-N content in the LP and LC groups was consistently lower than that in the CK and EF groups during ensiling. In first 7 days, the number of LAB significantly increased and then stabilized. Except for the CK group, other groups of yeast were inhibited on the 7th day and then remained below the detection limit. Aerobic bacteria are only significantly affected by ensiling time (p < 0.05) and are inhibited 7 days before ensiling, while their numbers remain around 10³ cfu/g.

Table 1

Fermentation quality of TMR during ensiling
Item	Treatment	Ensiling time(d)					SEM	p-value
Item	Treatment	0	7	14	28	56	SEM	S	T	SⅩT
pH	CK	6.72^Aa	5.00^Ba	4.90^Ca	4.63^Db	4.55^Ea	0.105	< 0.001	< 0.001	< 0.001
	LP	6.75^Aa	4.93^Bb	4.79^Cb	4.58^Dc	4.52^Eb
	LC	6.68^Aa	4.91^Bc	4.78^Cb	4.54^Dd	4.50^Dc
	EF	6.69^Aa	4.99^Ba	4.90^Ca	4.65^Da	4.54^Ea
Lactic acid (g/kg DM)	CK	10.8^Da	51.8^Cb	58.1^Bab	69.2^Aa	73.9^Aa	3.011	< 0.001	< 0.001	0.055
	LP	9.8^Da	55.1^Cab	63.8^Bab	71.2^Aa	74.5^Aa
	LC	9.4^Da	57.1^Ca	64.3^Ba	72.2^Aa	75.2^Aa
	EF	10.4^Ca	52.5^Bb	57.1^Bb	68.4^Aa	72.6^Aa
Acetic acid (g/kg DM)	CK	0^Bb	6.90^Aa	6.41^Aa	6.19^Aa	7.06^Aa	0.241	0.016	< 0.001	< 0.001
	LP	2.32^Ca	5.76^Ba	5.97^ABa	6.31^Aa	6.36^Aab
	LC	2.53^Ca	5.89^ABa	5.95^ABa	6.11^Aa	5.64^Bb
	EF	2.98^Ba	6.44^Aa	6.27^Aa	6.30^Aa	6.97^Aa
Propionic acid (g/kg DM)	CK	ND	ND	ND	ND	1.90	-	-	-	-
	LP	ND	ND	ND	1.88	1.88
	LC	ND	ND	ND	1.85	1.87
	EF	ND	ND	0.61	1.85	1.92
Butyric acid (g/kg DM)	CK	ND	ND	ND	ND	ND	-	-	-	-
	LP	ND	ND	ND	ND	ND
	LC	ND	ND	ND	ND	ND
	EF	ND	ND	ND	ND	ND
NH₃-N (% TN)	CK	0.74^Da	1.82^Ca	2.84^Ba	3.45^Aa	3.62^Aa	0.133	< 0.001	< 0.001	0.002
	LP	0.65^Da	1.41^Cc	2.19^Bb	2.92^Ab	3.15^Ab
	LC	0.64^Da	1.57^Cb	2.22^Bb	2.91^Ab	3.14^Ab
	EF	0.73^Da	1.84^Ca	2.77^Ba	3.41^Aa	3.59^Aa
DM, dry matter; TN, total nitrogen; S, LAB strains; T, ensiling time; SⅩT, Interaction between LAB and ensiling time;ND, not detected; SEM, standard error of the means; LP, L. plantarum; LC, L. casei; EF, E. faecium; CK, control; Means with different letters in the same column (a–c) differ (p < 0.05); Means with different letters in the same row (A–E) differ (p < 0.05).

Table 2

Microbial composition during ensiling of TMR
Items	Treatment	Ensiling time(d)					SEM	p-value
Items	Treatment	0	7	14	28	56	SEM	S	T	SⅩT
LAB (log₁₀ cfu/g FW)	CK	6.51^Db	8.85^Aa	7.98^Ca	8.23^BCa	8.52^ABab	0.080	< 0.001	< 0.001	< 0.001
	LP	7.48^Ca	8.94^Aa	8.16^Ba	8.18^Ba	8.34^Bb
	LC	7.44^Ca	8.96^Aa	8.28^Ba	8.28^Ba	8.68^ABa
	EF	7.31^Ca	8.86^Aa	7.99^Ba	8.14^Ba	8.39^ABb
Yeast (log₁₀ cfu/g FW)	CK	3.92	2.5	ND	ND	ND	-	-	-	-
	LP	3.75	ND	ND	ND	ND
	LC	3.89	ND	ND	ND	ND
	EF	3.87	ND	ND	ND	ND
Aerobic bacteria (log₁₀ cfu/g FW)	CK	7.33^Aa	3.28^Ba	3.27^Ba	3.78^Ba	3.24^Ba	0.218	0.173	< 0.001	0.538
	LP	7.06^Aa	3.16^Ba	3.06^Ba	3.56^Ba	2.76^Ba
	LC	7.53^Aa	2.80^Ca	2.96^Ca	3.44^Ba	3.25^BCa
	EF	7.29^Aa	2.94^Ca	3.15^Ca	3.79^Ba	3.03^Ca
FW, fresh weight; S, LAB strains; T, ensiling time; SⅩT, Interaction between LAB and ensiling time; ND, not detected; SEM, standard error of the means; LP, L. plantarum; LC, L. casei; EF, E. faecium; CK, control; Means with different letters in the same column (a–c) differ (p < 0.05); Means with different letters in the same row (A–E) differ (p < 0.05).

3.2 Chemical composition of TMR silage

The impact of various LAB strains on the chemical composition of TMR silage throughout the ensiling process is presented in Table 3. The type of LAB, ensiling time, and their interaction have a significant impact on the content of WSC, CP, NDF, and ADF (p < 0.05), while the content of DM is only affected by ensiling time (p < 0.001). With the extension of ensiling time, except for the LC and EF groups, the DM content significantly decreased, while the LP group had the largest drop in DM content. The WSC content of all TMR silages decreased rapidly in the first 7 days. After that, the decline was slower. After 56 days of ensiling, the CP and ADF contents of all TMR silages increased slightly, and the NDF and ADF contents of the CK, LP and LC groups also increased slightly, but the NDF content of the EF group decreased slightly.

Table 3

Chemical composition of TMR during ensiling
Items	Treatment	Ensiling time(d)					SEM	p-value
Items	Treatment	0	7	14	28	56	SEM	S	T	SⅩT
DM (g/kg FW)	CK	581^Aa	568^ABa	563^Ba	565^ABa	566^ABa	1.357	0.072	< 0.001	0.955
	LP	576^Aa	571^ABa	557^ABa	556^ABa	553^Bb
	LC	580^Aa	574^Aa	568^Aa	564^Aa	565^Aa
	EF	574^Aa	565^Aa	560^Aa	560^Aa	563^Aa
WSC (g/kg DM)	CK	99.7^Aa	69.3^Ba	65.9^Bb	62.0^BCb	54.5^Cb	2.570	< 0.001	< 0.001	< 0.001
	LP	100.4^Aa	62.2^Ba	55.6^BCc	48.3^BCc	43.4^Cc
	LC	109.6^Aa	60.7^Ba	55.9^Bc	47.1^Cc	43.5^Cc
	EF	99.2^Aa	65.0^Ba	77.3^Ba	72.5^Ba	74.0^Ba
CP (g/kg DM)	CK	164^Cb	164^Cb	181^Aab	174^Bb	181^Ab	0.090	< 0.001	< 0.001	< 0.001
	LP	166^Db	175^Ca	185^ABa	178^BCab	188^Aa
	LC	178^ABa	172^Bab	183^Aab	181^Aa	182^Ab
	EF	179^ABa	179^Ba	178^Bb	177^Bab	185^Aab
NDF (g/kg DM)	CK	286^Aa	270^Aa	276^Ab	245^Ba	286^Aa	1.858	0.001	0.003	< 0.001
	LP	263^CDb	278^ABa	254^Dc	268^BCa	284^Aa
	LC	263^Ab	278^Aa	286^Aa	283^Aa	271^Aab
	EF	271^ABb	260^BCb	282^Aab	248^Da	256^CDb
ADF (g/kg DM)	CK	202^Aa	188^Bb	192^Ba	173^Ca	202^Aa	1.612	< 0.001	< 0.001	< 0.001
	LP	176^Cc	191^ABb	172^Cb	188^Ba	198^Aa
	LC	183^Ab	208^Aa	191^Aa	200^Aa	202^Aa
	EF	184^Bb	177^Cb	195^Aa	170^Da	183^Bb
DM, dry matter; FW, fresh weight; WSC, water soluble carbohydrates; CP, crude protein; NDF, neutral detergent fiber; ADF, acid detergent fiber; S, LAB strains; T, ensiling time; SⅩT, Interaction between LAB and ensiling time; ND, not detected; SEM, standard error of the means; LP, L. plantarum; LC, L. casei; EF, E. faecium; CK, control; Means with different letters in the same column (a–c) differ (p < 0.05); Means with different letters in the same row (A–D) differ (p < 0.05).

3.3 Vitamin A changes in TMR silage with different LAB inoculations

As shown in Fig. 1, there is a significant difference (p < 0.05) in the effect of different LABs on vitamin A loss during TMR ensiling. After 56 days of ensiling, the LC group exhibits the highest loss rate of vitamin A, reaching 58.9%. The LP group, on the other hand, has the lowest loss rate of vitamin A at 44.3%. In the CK and EF groups, the vitamin A loss rates were 51.3% and 47.5%, respectively. Therefore, inoculation with LP and EF reduced the loss of vitamin A in TMR silage, while inoculation with LC did not reduce the loss of vitamin A.

3.4 The tolerance of LAB to H₂O₂

The tolerance of different LAB to H₂O₂ is shown in Fig. 2a. Under the condition of adding 2 mM H₂O₂, the growth of LC, EF, and LP was inhibited. After 24h of cultivation, the OD600 values of LC, EF, and LP were 1.1, 0.86, and 0.75, respectively. Further analysis showed that LC, EF and LP differed significantly in tolerance to H₂O₂ (p < 0.05). Among them, LP exhibited the strongest tolerance to H₂O₂, followed by LC, and EF showed the weakest tolerance to H₂O₂.

3.5 DPPH radical scavenging activity

As shown in Fig. 2b, this study measured the scavenging activity of the fermentation broth, cells, and cell-free extracts of LC, EF, and LP on DPPH radicals. The results demonstrated that the fermentation broth, cells, and cell-free extracts of LC, EF, and LP all exhibited scavenging activity against DPPH radicals. However, significant differences in scavenging capacity were observed (p < 0.05). The highest DPPH radical scavenging activity was observed in the fermentation broth of LP, LC, and EF (85%, 79%, and 62%, respectively), followed by the cells with scavenging capacities of 40%, 38%, and 26%, respectively. The weakest scavenging activity against DPPH radicals was observed in the cell-free extracts, with scavenging capacities of 16%, 12%, and 10%, respectively.

Furthermore, a comparison of the scavenging rates of the same components of LP, LC, and EF against DPPH radicals revealed significant differences (p < 0.05) among the three strains. Among them, LP exhibited the strongest scavenging activity against DPPH radicals in all components, with LC demonstrating intermediate scavenging activity and EF exhibiting the weakest scavenging activity against DPPH radicals.

3.6 Hydroxyl Radical (●OH) Scavenging Ability

Figure 2c illustrates that three strains of LAB possess scavenging abilities against ●OH radicals. However, there are notable differences between them. It was observed that there were significant differences among the components of the same LAB and among the same components of different LAB (p < 0.05). Compared to the fermentation broth and cells, the cell-free extracts of LP, LC, and EF exhibited stronger ●OH scavenging abilities. Among them, the cell-free extract of EF demonstrated the highest capability in scavenging ●OH radicals. Furthermore, significant differences were observed in the ●OH scavenging abilities among the fermentation broth and cell components of each strain. The order of scavenging abilities in the fermentation broth was EF > LC > LP, while the same order was observed in the cells.

3.7 Superoxide Radical (O²⁻) Scavenging Ability Determination

As depicted in Fig. 2d, all components of the three LAB strains exhibited O²⁻ scavenging abilities. The fermentation broth of LP showed the strongest capability in scavenging O²⁻ radicals at 70.3%. Significant differences were observed in the O²⁻ scavenging abilities among the various constituent parts of the same LAB strain, with the cells and cell-free extracts demonstrating reduced capabilities compared to the fermentation broth. Significant discrepancies were also identified in the O²⁻ scavenging capabilities among the same components of disparate LAB (p < 0.05). Among the cells, LC exhibited the strongest O²⁻ scavenging ability, while among the cell-free extracts, LP showed the strongest O²⁻ scavenging ability.

3.8 Antioxidant Activity of LAB

This study conducted a comparative analysis of the total antioxidant capacity (T-AOC) and the enzymatic activities of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and catalase (CAT) among the LP, LC, and EF groups. These results are displayed in Table 4. It was observed that the T-AOC levels in the fermentation broth of all three LAB were higher than in their cells and cell-free extracts. The LP fermentation broth exhibited the highest T-AOC level. All three LAB exhibited antioxidant enzyme activities. The fermentation broth of LP exhibited the highest SOD activity, the cell exhibited the highest GSH-Px activity and the cell-free extract exhibited the highest CAT activity. However, the cell-free extract exhibited the lowest SOD activity. In summary, the antioxidant activity of LAB was ranked from highest to lowest as follows: LP > LC > EF.

Table 4

T-AOC, SOD, GSH Px and CAT activities of LAB
Strain	Fermentation broth(U/mL)				Cells(U/mL)				Cell-free extracts(U/mL)
Strain	T-AOC	SOD	GSH-Px	CAT	T-AOC	SOD	GSH-Px	CAT	T-AOC	SOD	GSH-Px	CAT
LP	38.87	57.64	19.35	0.41	1.54	16.52	11.84	2.54	0.78	9.89	12.75	2.38
LC	30.69	42.88	14.56	0.21	0.94	10.37	5.63	0.89	0.84	7.25	9.47	0.86
EF	21.32	36.36	10.35	0.34	0.38	7.59	2.43	0.96	0.34	2.54	5.58	0.36
LP, L. plantarum; LC, L. casei; EF, E. faecium; T-AOC, total antioxidant capacity; SOD, superoxide dismutase; GSH-Px, glutathione peroxidase; CAT, catalase.

3.9 Antioxidant Activity of Fermented TMR

As indicated in Table 5, after a 56-day ensiling duration of TMR inoculated with various strains of LAB, significant disparities in antioxidant activity were identified among the treatment groups (p < 0.05). The T-AOC values of TMR fermented with LP and LC were found to be significantly higher than those of the CK and EF groups. The highest T-AOC value was observed in the LP group. However, the SOD activity in TMR fermented with LP and LC was found to be lower than that in the CK and EF groups. The GSH-Px and CAT activities in the TMR fermented with LAB were higher than those in the CK, with the highest GSH-Px and CAT activities observed in the TMR fermented with LP. Consequently, it can be stated that the antioxidant activity of the TMR fermented with LP is greater than that of the TMR fermented with the other two LAB.

Table 5

Antioxidant activity of TMR after 56 days of ensiling
Items	CK	LP	LC	EF	SEM	p-value
T-AOC, U/g FW	265^C	307^A	289^B	274^C	4.842	< 0.001
SOD, U/g FW	489^A	456^B	454^B	481^A	4.902	0.001
GSH-Px, U/g FW	1432^D	1992^A	1725^B	1689^C	5.930	< 0.001
CAT, U/g FW	18.4^D	42.2^A	34.6^B	23.9^C	0.746	< 0.001
LP, L. plantarum; LC, L. casei; EF, E. faecium; T-AOC, total antioxidant capacity; SOD, superoxide dismutase; GSH-Px, glutathione peroxidase; CAT, catalase; FW, Fresh matter; SEM, standard error of the means; Means with different letters in the same row (A–D) differ (p < 0.05).

4.1 The impact of LAB on the fermentation quality and chemical composition of TMR silage

This study explored the influence of incorporating various strains of Lactic Acid Bacteria (LAB) on the fermentation traits of Total Mixed Rations (TMR) as depicted in Tables 1 and 2. Upon completion of the 56-day ensiling phase, the pH values across all treatment groups were confined within the narrow range of 4.50 to 4.55. These findings align with the outcomes reported by Chen et al. (2015), which indicated that at elevated DM concentrations exceeding 40%, the pH typically fluctuated between 4.0 and 5.0 [31]. Furthermore, Nishino and Hattori (2007) reported that the pH decreased to 4.09 after 60 days of ensiling of TMR silage supplemented with LC [22]. However, in this study, the pH of the TMR in the LC group only decreased to 4.50 after 56 days of ensiling. This outcome is potentially attributable to the variations in the constituent composition of the TMR ingredients. LP, a homofermentative LAB, is known to promote acid production during ensiling, resulting in significantly lower pH values throughout the fermentation process compared to the CK and EF groups. The pH values of CK and EF groups showed no significant differences during ensiling, indicating that EF had no significant effect on the pH variation of TMR silage. It has been reported that LP and LC can increase lactic acid content while reducing acetic acid content [23], and this study obtained the same results. Moreover, LP and LC were also observed to reduce the propionic acid content of TMR.

The NH₃-N content of TMR silage in each group is relatively low, with all values below 5% TN. This may be attributed to the higher DM content (> 40%) inhibiting the growth of clostridium, as NH₃-N is mainly generated by the metabolism of amino acids by clostridium [24]. Additionally, butyric acid content was not detected in any group, further confirming the inhibition of clostridia fermentation. The NH₃-N content in the CK group was found to be higher than that in the LP and LC groups. This may be attributed to the slower pH decline observed in the CK group, which has been shown to favour the hydrolytic action of microbial proteases. In this experiment, the addition of LP, LC, and EF did not significantly affect the quantity of LAB and aerobic bacteria. However, they were found to rapidly inhibit yeast growth on the 7th day. This finding is consistent with the findings of Jiang et al. (2020), who reported that yeast was below the detection limit after 14 days of fermentation with LP-inoculated TMR [10]. This indicates that inoculating LAB in TMR is unfavorable for yeast growth.

The impact of LAB on DM content was not statistically significant in this experiment, with a loss of less than 2.3%. The LP and LC groups exhibited a significant WSC loss, while the WSC content in EF demonstrated an upward trend on the 14th day. This may be attributed to EF's ability to degrade starch into soluble sugars in the later stages of fermentation, as previously described in reference [12].

After 56 days of ensiling, the loss of DM led to an increase in CP, NDF and ADF. However, the reduction in NDF content in the EF group may be due to the ability of EF to degrade certain components of NDF.

4.2 Vitamin A content influenced by LAB

Based on the results of the previous research, LAB may be correlated with the loss of vitamin A [6]. This study investigated the influence of different LAB strains on vitamin A in TMR silage. The results showed that the rate of vitamin A loss was in descending order: LC > CK > EF > LP (Fig. 1). Previous studies have indicated that a reduction in pH can expedite the degradation of vitamin A [7]. In the current investigation, the pH levels of both the LP and LC groups experienced a parallel decline. Consequently, one would anticipate no significant divergence in vitamin A degradation between these two groups. However, the rate of vitamin A loss in the LC group was substantially higher compared to that in the LP group, as illustrated in Fig. 1. Similarly, no significant variance in pH levels was observed between the EF group and CK group throughout the ensiling process. However, the rate of vitamin A degradation in the CK group markedly exceeded that of the EF group. Consequently, these findings imply that, in addition to pH, there must be other factors (caused by LAB) that can affect the changes in vitamin A content. Studies by Siedler et al. (1956) and Kurzer et al. (2014) have shown that the addition of antioxidants to skim milk or poultry feed can improve its antioxidant properties, thereby reducing vitamin A loss [25–26]. Likewise, Feng and Wang (2020) have shown that LAB can produce antioxidant enzymes and therefore have certain antioxidant properties [27]. Therefore, we speculate that different LAB may have different effects on the antioxidant capacity of TMR silage. This may result in differences in vitamin A loss between different groups.

4.3 Antioxidant activity of LAB

The effect of the antioxidant activity of LAB on the TMR silage was investigated. The results demonstrated that LP exhibited a higher scavenging capacity for DPPH radicals and O²⁻radicals, as well as a higher tolerance for H₂O₂ (Fig. 2). These findings indicate that LP has the strongest ability to scavenge free radicals. Furthermore, a multitude of experiments have demonstrated that LAB possesses antioxidant activity. The mechanism by which it exerts antioxidant effects may be related to its ability to produce a variety of antioxidant enzymes, including SOD, GSH-Px and CAT [28]. In this experiment, the SOD, GSH-Px and CAT activities of all fractions of LP were found to be higher than those of LC and EF (Table 4). This may be responsible for the higher T-AOC of LP. While LC exhibited higher T-AOC and SOD activities in all fractions than EF, the GSH-Px activities of fermentation broth and cells were slightly lower than those of EF. Therefore, in a comprehensive view, the antioxidant activity of LC was higher than that of EF. Consequently, the antioxidant activities of LAB in descending order were LP > LC > EF.

4.4 Antioxidant activity of TMR silage influenced by LAB

The antioxidant capacity of TMR silage inoculated with LAB was also determined (Table 5). The results showed that different LAB improved the antioxidant capacity of TMR silage differently. The most elevated levels of T-AOC and activities of GSH-Px, as well as CAT, were detected in the Low-Protein (LP) group, succeeded by the LC group, and ultimately the EF group. In contrast, the SOD activity in the TMR silage of the LP and LC groups was notably reduced in comparison to the CK and EF groups. Nonetheless, the T-AOC of the TMR silage in the LP and LC groups was significantly superior to that of the CK and EF groups. This suggests that the enhancement of the antioxidant capacity of TMR silage was mainly due to the role of GSH-Px and CAT.

4.5 The impact of antioxidant activity on vitamin A in relation to LAB

Based on the results of 4.2, the loss of vitamin A caused by inoculation of different LAB in TMR was variable. Inoculation with LP and EF reduced the loss of vitamin A in TMR silage, whereas inoculation with LC did not reduce vitamin A loss. This discrepancy may be attributed to the differing fermentation characteristics of TMR influenced by LAB, and the varying effects of LAB on the antioxidant activity of TMR silage. TMR silage of the LP group exhibited the highest T-AOC and GSH-Px activities and CAT activities. This suggests that the inoculation of LP may have enhanced the antioxidant capacity of TMR silage, thereby reducing vitamin A loss. The SOD activity of the TMR silage in the LP and LC groups was found to be lower than that of the CK and EF groups. This can be attributed to the fact that the main role of SOD is to catalyse the conversion of O²⁻ to H₂O₂, and then GSH-Px and CAT decompose H₂O₂ to H₂O by donating electrons to H₂O₂ and ●OH [26, 32]. However, it is important to note that O²⁻ is produced in electron transfer reactions during the transport of oxygen in alkaline and aerobic environments [29–30]. This suggests that the lower pH of TMR silage in the LP and LC groups may have inhibited SOD activity. Additionally, although the T-AOC of TMR silage in the LC group was higher than that of EF and CK, the loss of vitamin A was greater. Previous study has indicated that vitamin A is sensitive to acidic conditions [7]. Therefore, the reason for presenting this result may be due to the lower pH of TMR silage in the LC group, and the active capacity of GSH-Px and CAT in the LC group was not sufficient to resist the loss of vitamin A caused by the decrease in pH. Consequently, inoculation with LC did not result in a reduction in the loss of vitamin A from TMR silage. The T-AOC, GSH-Px and CAT activities of fermented TMR in the EF group were higher than that of the CK group, while the level of decrease in pH was comparable to that of the CK group. Therefore, this may be the reason for the lower loss of vitamin A from fermented TMR in the EF group compared to that of the CK group.

The fermentation quality of TMR inoculated with LP and LC was significantly superior to that of the CK group, whereas the fermentation quality of TMR inoculated with EF was not significantly different compared to the CK group. The loss of vitamin A in TMR silage was found to vary across the groups, with LC exhibiting the greatest loss, followed by CK, EF, and LP. In addition, an analysis of the antioxidant capacity of different LAB and its effect on vitamin A loss in TMR silage revealed a correlation between the strength of the antioxidant capacity of the LAB and the loss of vitamin A. Inoculation of LAB with a higher antioxidant capacity could significantly increase the antioxidant capacity of TMR silage, thus reducing the loss of vitamin A. However, when the pH drop was severe, the enhancement of antioxidant capacity might also fail to reduce vitamin A loss.

Author Contribution

Yumeng Cheng: Experiment investigation, writing-Original draft preparation, sampling and analysis. Zidie Wu: Data curation, formal analysis. Kaijian Bi: Investigation, validation. Haizhong Yu: Resources, Software. Xiqing Wang and Pengjiao Tian: Writing- Reviewing and Editing, Project administration, Funding acquisition.

Acknowledgement

This study support by the International Science and Technology Cooperation Project of Hubei Province (2023EHA051), Hubei Provincial Natural Science Foundation Program (2024AFC001), Hubei Province College Student Innovation and Entrepreneurship Project (S202310519038), National Natural Science Foundation of China Cultivation Project (2023pygqzk09) and Hubei University of Arts and Science Teacher Research Ability Launch Project (kyqdf2021011).

Data Availability

The authors declare that the data supporting the findings of this study are available within the paper. Should any raw datasets are available from the corresponding author upon reasonable request.

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No competing interests reported.

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Vitamin A in total mixed ration silage influenced by various lactic acid bacteria: Lactobacillus plantarum, Lactobacillus casei and Enterococcus faecium

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Abstract

Figures

1. Introduction

2. Material and Methods

2.1 LAB activated

2.2 TMR and TMR silage preparation

2.3 Chemical Analysis

2.4 Microbial Analysis

2.5 LAB antioxidant activity determination

2.5.1 Preparation of cells and intracellular cell-free extracts

2.5.2 Scavenging of DPPH free radical

2.5.3 Scavenging of hydroxyl radical (•OH)

2.5.4 Superoxide anion (O2−) scavenging ability

2.5.5 Tolerance to hydrogen peroxide

2.5.6 Determination of antioxidant activity of LAB

2.5.7 Determination of antioxidant activity of TMR fermentation

2.6 Statistical Analysis

3. Results

3.1 Fermentation quality and microbial composition of TMR

3.2 Chemical composition of TMR silage

3.3 Vitamin A changes in TMR silage with different LAB inoculations

3.4 The tolerance of LAB to H2O2

3.5 DPPH radical scavenging activity

3.6 Hydroxyl Radical (●OH) Scavenging Ability

3.7 Superoxide Radical (O2−) Scavenging Ability Determination

3.8 Antioxidant Activity of LAB

3.9 Antioxidant Activity of Fermented TMR

4. Discussion

4.1 The impact of LAB on the fermentation quality and chemical composition of TMR silage

4.2 Vitamin A content influenced by LAB

4.3 Antioxidant activity of LAB

4.4 Antioxidant activity of TMR silage influenced by LAB

4.5 The impact of antioxidant activity on vitamin A in relation to LAB

5. Conclusion

Declarations

Author Contribution

Acknowledgement

Data Availability

References

Additional Declarations

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2.5.4 Superoxide anion (O²⁻) scavenging ability

3.4 The tolerance of LAB to H₂O₂

3.7 Superoxide Radical (O²⁻) Scavenging Ability Determination