Lactic acid bacterial inoculant effects on the vitamin content of alfalfa and Chinese leymus silage

Objective Information regarding the vitamin content of silage is limited. This study investigated the changes in the vitamin content of alfalfa and Chinese leymus silages with or without a lactic acid bacterial inoculant. Methods Alfalfa at the early flowering stage and Chinese leymus at the full-bloom stage were harvested. The treatments for each forage type were control (deionized water only) and 1×106 colony-forming units Lactobacillus plantarum (LP)/g fresh matter. After 45 days of ensiling, all silages were sampled for evaluating the vitamin content, fermentation quality and chemical composition. Results The LP inoculant decreased the pH value and ammonia nitrogen content of the alfalfa and Chinese leymus silages and significantly (p<0.05) increased the lactic acid, acetic acid concentrations and Flieg’s points. Prior to ensiling, the levels of five B-group vitamins (thiamin, riboflavin, niacin, pantothenic acid, and pyridoxine) and α-tocopherol in alfalfa were significantly (p<0.01) higher than those in Chinese leymus. Ensiling decreased the levels of the five B-group vitamins in both alfalfa and Chinese leymus while increasing the α-tocopherol content of Chinese leymus. The thiamin, riboflavin, niacin and pantothenic acid levels in the LP-treated silage were significantly (p<0.05) lower than those in the untreated silage for the alfalfa and Chinese leymus. The α-tocopherol content in the LP-treated alfalfa silage was significantly (p<0.05) higher than that in the untreated alfalfa silage. There was no significant (p>0.05) difference in pyridoxine content between the untreated and LP-treated silages for both forages. Conclusion With or without LP inoculation, the levels of the five B-group vitamins (thiamin, riboflavin, niacin, pantothenic acid, and pyridoxine) in alfalfa and Chinese leymus decreased after 45 days of ensiling, while the α-tocopherol content of Chinese leymus increased. The LP inoculant improved the fermentation quality of both the alfalfa and Chinese leymus silages but increased the thiamin, riboflavin, niacin, and pantothenic acid loss in the two forages after fermentation.


INTRODUCTION
Vitamins are organic nutrients that are essential in minute quantities for the nutrition of ruminants [1]. Vitamins act as coenzymes and precursors of coenzymes in the regulation of metabolic processes [2]. Fresh or conserved forages are important dietary sources of natural vitamins in ruminants [1,3]. The concentrate used in ruminant diets does not contain any natural vitamins or vitamin precursors [4]. αTocopherol and βcarotene in forage have received much attention due to their antioxidant properties [3,59]. The level of αtocopherol in ensiled forage was seen to be higher than that in hay [6,7]. There is little information regarding the levels of Bgroup vitamins in silage and the effects of additives on the B vitamin content. Bgroup vitamins are essential for ruminants, and vitamin B deficiency can lead to different degrees of metabolic disorders [7]. It has been shown that there is an increasing need for B vitamins in ru minants due to the increased demand for high productivity and quality of animal products [4]. However, to the best of our knowledge, there have been few studies on Bgroup vita mins in silage. Additional information is required regarding the changes in B vitamin levels in silage.
Alfalfa and Chinese leymus are the main forage sources for animal diets in China. Storage of this forage as silage is a good way to retain the nutritional content. Additives are added dur ing forage ensilage to achieve highquality silage. The additives strongly influence the fermentation process of silage and may influence the vitamin content in silage [7]. Shingfield et al [8] demonstrated an increase in the αtocopherol content in si lage via the use of an inoculant enzyme preparation. Liu et al [9] reported that the use of additives decreased the αtocopherol and βcarotene levels in silages. However, there are no reports in the literature on the effect of lactic acid bacterial additives on Bgroup vitamins in silage. Certel et al [10] and Ochanda et al [11] found that fermentation influences the concen trations of Bgroup vitamins in food. However, information regarding Bgroup vitamins in silage is limited. Therefore, this study was undertaken to provide information on the ef fects of lactic acid bacterial inoculants on the vitamin content of alfalfa and Chinese leymus silage. Additionally, the fermen tation quality and chemical composition of alfalfa and Chinese leymus silage were also evaluated.

Silage materials and ensiling
Alfalfa and Chinese leymus were grown in Guyuan County (41°42′41°57′N, 115°32′115°59′E), Hebei Province, P. R. China. Alfalfa at the early flowering stage and Chinese leymus at the fullbloom stage were harvested with 4 to 5cm stubble height and wilted for 2 hours. Then, the alfalfa and Chinese leymus forages were chopped into 1 to 2cm pieces with a for age cutter. The treatments for each forage type were control (deionized water only) and Lactobacillus plantarum inoculant (LP). The lactic acid bacterial strain LP was isolated from the grass and identified as L. plantarum via 16S rDNA sequencing. The LP strain was inoculated at 1×10 6 colonyforming units/g fresh matter. Approximately 300 g of chopped alfalfa and Chinese leymus forages were filled into polyethylene bag silos (30×40 cm; 0.19 mm thickness; 50 cm 3 m -2 day -1 0.1 Mpa -1 ). Each treatment had three repetitions. The silos were sealed with a vacuum sealer (FW3150; Fresh World Electric Co., Ltd., Guangzhou, China). All the silos were preserved at ambient temperature in a dark room, then opened and sampled after ensiling for 45 days.

Chemical analysis
Twentygram samples of alfalfa and Chinese leymus silages were mixed with 180 mL of distilled water and then homog enized in a juicer for 2 min. The mixture was filtered, and then, the filtrate was used for determination of pH and organic acid and ammonia nitrogen concentrations. The pH was measured by using a pH meter (PHS3C, INESA Scientific Instrument Co., Ltd., Shanghai, China). The organic acid (lactic, acetic, propionic and butyric acids) levels were determined by high performance liquid chromatography (HPLC; column: Shodex RS Pak KC811, Showa Denko KK, Kawasaki, Japan; detector: DAD, 210 nm, SPD20A, Shimadzu Co., Ltd., Kyoto, Japan; eluent: 3 mmol/L HClO 4 , at a flow rate of 1.0 mL/min; column temperature: 50°C). The ammonia nitrogen concentration was analyzed by the phenol and sodium hypochlorite method [12]. The buffering capacity of the alfalfa and Chinese leymus materials was determined by the method described by Playne and McDonald [13]. The dry matter (DM) content was mea sured after ovendrying at 65°C for 48 hours. The ovendried samples were first milled and passed through a 1.0mm screen and then used for analysis of the watersoluble carbohydrate (WSC), crude protein (CP), neutral detergent fiber (NDF), acid detergent fiber (ADF), and hemicellulose (HC) levels. The WSC content was measured by the anthrone method [14]. The CP content was analyzed by using method 976.05 of the Association of Official Analytical Chemists [15]. The NDF and ADF levels were measured by the method described by Van Soest et al [16]. The HC content of the samples was estimated as the NDF value minus the ADF value.

Vitamin analysis
The vitamin concentrations were determined by HPLC. The sample (100 g) was freeze dried using a vacuum freezedry ing machine for 2 days (FreeZone 4.5L, LABCONCO Corp., Kansas City, MO, USA) before analysis. The freezedried sam ples were then ground and passed through a 1.0mm screen.
Thiamin concentrations were determined in duplicate based on the method of GB/T 147002018. The extraction solution was prepared as follows: 107 g of NH 4 Cl was dissolved in 1,000 mL of ultrapure water; then, the pH was adjusted to 3 to 4 with 2 mol/L HCl; 900 mL of this NH 4 Cl solution was mixed with 100 mL of carbinol (SigmaAldrich, Darmstadt, Germany). Then, 5 g of sample and 70 mL of extraction solution were mixed in a 100mL brownglass volumetric flask. Next, the samples were incubated in an ultrasonic bath (30 min). After cooling in an ice bath, the volume was made up to 100 mL, and the mixture was centrifuged (8,000 r/min, 5 min). The supernatant was collected and passed through a 0.45μm syringe filter. Twenty microliters of the filtered supernatant were injected into an HPLC instrument. The samples were examined on a Pursuit 5μm C18 column (150×4.6 mm; Agilent, Ammerbuch, Germany). The mobile phase used was prepared as follows: 1.1 g of sodium heptanesulfonate and 50 mg of disodium ethylenediamine tetraacetic acid were dissolved in 700 mL of ultrapure water, and after total disso lution, 25 mL of glacial acetic acid and 10 mL of triethylamine (SigmaAldrich, Germany) were added. Then, the solution was made up to 1,000 mL, and the pH value was adjusted to 3.7. Then, 800 mL of this solution was mixed with 200 mL of carbinol (SigmaAldrich, Germany). The flow rate was 1.0 mL/min, and the diode array detector was operated at a wavelength of 242 nm. The column temperature was 25°C to 28°C.
Riboflavin levels were analyzed in duplicate according to the method of GB/T 147012002. The extraction solution was prepared as follows: 50 mg of disodium ethylenediamine tetraacetic acid was dissolved in 700 mL of ultrapure water, and after total dissolution, 25 mL of glacial acetic acid and 5 mL of triethylamine were added, and the volume was ad justed to 1,000 mL. Then, 5 g of sample and 30 mL of extraction solution were mixed in a 100mL brownglass volumetric flask. Next, the samples were incubated in an 80°C to 100°C water bath (30 min). After cooling in an ice bath, 14 mL of carbinol was added. Then, the volume was made up to 100 mL with extraction solution, and the solution was centri fuged (8,000 r/min, 5 min). The supernatant was collected and passed through a 0.45μm syringe filter. Then, 10 μL of the filtered supernatant was injected into an HPLC instru ment. The samples were examined on a Pursuit 5μm C18 column (150×4.6 mm; Agilent, Germany). The mobile phase was prepared as follows: 1.1 g of sodium heptanesulfonate and 50 mg of disodium ethylenediamine tetraacetic acid was dissolved in 700 mL of ultrapure water, and after total dissolution, 25 mL of glacial acetic acid and 5 mL of triethyl amine (SigmaAldrich, Germany) were added. The solution was then made up to 1,000 mL, and the pH value was ad justed to 3.4; 860 mL of this solution were mixed with 140 mL of carbinol (SigmaAldrich, Germany). The flow rate was 0.8 mL/min, and the diode array detector was operated at a wavelength of 280 nm. The column temperature was 25°C to 28°C.
Niacin levels were examined according to the method of GB/T 178132018. The extraction solution was prepared as follows: 50 mg of disodium ethylenediamine tetraacetic acid was dissolved in 800 mL of ultrapure water, and then, 20 mL of glacial acetic acid and 5 mL of triethylamine were added; after thorough mixing, this solution was mixed with 200 mL of carbinol (SigmaAldrich, Germany). Then, 5 g of sample, 1 g of disodium ethylenediamine tetraacetic acid and 70 mL of extraction solution were mixed in a 100mL brownglass volumetric flask. Next, the samples were incubated in an ultra sonic bath (15 min). After cooling in an ice bath, the volume was made up to 100 mL with extraction solution, and the mixture was centrifuged (8,000 r/min, 5 min). The superna tant was collected and passed through a 0.45μm syringe filter.
Then, 20 μL of the filtered supernatant was injected into an HPLC instrument. The samples were examined on a Pursuit 5μm C18 column (150×4.6 mm; Agilent, Germany). The mobile phase was prepared as follows: 1.1 g of sodium heptane sulfonate and 50 mg of disodium ethylenediamine tetraacetic acid were dissolved in 1,000 mL of ultrapure water, and after total dissolution, 20 mL of glacial acetic acid and 5 mL of triethylamine (SigmaAldrich, Germany) were added, and the pH value was adjusted to 4.0. Then, 800 mL of this solu tion was mixed with 200 mL of carbinol (SigmaAldrich, Germany). The flow rate was 1.0 mL/min, and the diode array detector was operated at a wavelength of 262 nm.
Pantothenic acid levels were measured based on the method of GB/T 183972014. Five grams of sample and 50 mL of the mobile phase were mixed in a 150mL conical flask. The sam ples were incubated in an ultrasonic bath (15 min). After cooling in an ice bath, the samples were centrifuged (8,000 r/min, 5 min). The supernatant was collected and passed through a 0.45μm syringe filter. Then, 10 μL of the filtered supernatant was injected into an HPLC instrument. The sam ples were examined on a Pursuit 5μm C18 column (150×4.6 mm; Agilent, Germany). The mobile phase was prepared by adding 50 mL of acetonitrile to 950 mL of 0.05% phosphoric acid (aqueous solution). The flow rate was 1.0 mL/min, and the diode array detector was operated at a wavelength of 200 nm.
Pyridoxine levels were determined based on the method of GB/T 147022002. The extraction solution was prepared as follows: 50 mg of disodium ethylenediamine tetraacetic acid was dissolved in 700 mL of ultrapure water, and then, 25 mL of glacial acetic acid and 5 mL of triethylamine were added, and the volume was adjusted to 1,000 mL. Then, 800 mL of this solution was mixed with 200 mL of carbinol (Sigma Aldrich, Germany). Then, 5 g of sample and 70 mL of sodium dihydrogen phosphate solution were mixed in a 100mL brownglass volumetric flask. Next, the samples were incu bated in an ultrasonic bath (20 min). After cooling in an ice bath, the volume was made up to 100 mL with extraction solution, and the mixture was centrifuged (8,000 r/min, 5 min). The supernatant was collected and passed through a 0.45μm syringe filter. Then, 20 μL of filtered supernatant was injected into an HPLC instrument. The samples were examined on a Pursuit 5μm C18 column (250×4.6 mm; Agilent, Germany). The mobile phase was prepared as fol lows: 1.1 g of sodium heptanesulfonate and 50 mg of disodium ethylenediamine tetraacetic acid were dissolved in 700 mL of ultrapure water, and after total dissolution, 25 mL of glacial acetic acid and 5 mL of triethylamine (SigmaAldrich, Ger many) were added, and the volume was adjusted to 1,000 mL with ultrapure water. The pH was adjusted to 4.0. Then, 800 mL of this solution was mixed with 200 mL of carbinol (Sig maAldrich, Germany). The flow rate was 1.0 mL/min, and the diode array detector was operated at a wavelength of 290 nm.
The αtocopherol levels were analyzed based on the method of GB/T 178122008. The extraction solution was prepared as follows: 50 mg of disodium ethylenediamine tetraacetic acid was dissolved in 700 mL of ultrapure water, and then, 25 mL of glacial acetic acid and 5 mL of triethylamine were added, and the volume was adjusted to 1,000 mL. Then, 800 mL of this solution was mixed with 200 mL of carbinol (Sig maAldrich, Germany). Then, 5 g of sample and 80 mL of carbinol were mixed in a 100mL brownglass volumetric flask. Next, the samples were incubated in an ultrasonic bath (60°C, 20 min). After cooling in an ice bath, the volume was made up to 100 mL with carbinol, and the solution was cen trifuged (8,000 r/min, 5 min). The supernatant was collected and passed through a 0.45μm syringe filter. Then, 20 μL of the filtered supernatant was injected into and HPLC instru ment. The samples were examined on a Pursuit 5μm C18 column (250×4.6 mm; Agilent, Germany). The mobile phase was composed of 98 mL of carbinol and 2 mL of ultrapure water. The flow rate was 1.0 mL/min, and the diode array de tector was operated at a wavelength of 285 nm.

Calculation of the Flieg's point
The quality of the alfalfa and Chinese leymus silage was esti mated based on the Flieg's point index, which was calculated by using the following equation [17]:

Statistical analysis
The vitamin content, fermentation quality, and chemical com position data were analyzed by analysis of variance (ANOVA) using the general linear modelunivariate procedure of SPSS 19.0 software [18]. ANOVAs were performed for forage types and treatments (control vs LP inoculant) as the two main para meters and for the interaction between the two parameters.
The mean values were compared using Duncan's multiple range tests. Differences between means were considered significant when p<0.05.

Chemical composition and vitamin content of alfalfa and Chinese leymus forages before ensiling
The chemical composition and vitamin content of the alfalfa and Chinese leymus forages before ensiling are summarized in Table 1. The DM levels in the alfalfa and Chinese leymus were 358.2 and 450.1 g/kg, respectively. The buffering capac ity of alfalfa was significantly (p<0.001) higher than that of Chinese leymus. The NDF, ADF, HC, and WSC levels in al falfa were significantly (p<0.01) lower than those in Chinese leymus. The CP level in alfalfa was significantly (p<0.001) higher than that in Chinese leymus. The thiamin, riboflavin, niacin, pantothenic acid, pyridoxine and αtocopherol con centrations in alfalfa were significantly (p<0.01) higher than those in Chinese leymus.

Fermentation characteristics of alfalfa and Chinese leymus silages
The fermentation characteristics of the alfalfa and Chinese leymus silages are presented in Table 2. The LP inoculant sig nificantly (p<0.05) decreased the pH values and significantly (p<0.05) increased the lactic acid and acetic acid levels of the alfalfa and Chinese leymus silages. For the Chinese leymus silage, the butyric acid levels and ratio of lactic acid levels to acetic acid levels in the LPtreated silage were significantly (p<0.05) lower those in the control. For the alfalfa silage, the ammonia nitrogen content was significantly (p<0.01) low ered by the LP inoculant. The pH value of the Chinese leymus silage was significantly (p<0.05) lower than that of the alfalfa silage, and the lactic acid, acetic acid and ammonia nitrogen levels in the Chinese leymus silage were significantly (p<0.05) lower than those in the alfalfa silage. The LP inoculants sig nificantly (p<0.05) increased the Flieg's points of the alfalfa and Chinese silages, and the Flieg's point of the Chinese ley mus silage was significantly (p<0.001) higher than that of the alfalfa silage.

Chemical compositions of alfalfa and Chinese leymus silages
The chemical compositions of the alfalfa and Chinese leymus silages are shown in Table 3. There was no significant differ ence (p>0.05) in DM, WSC, NDF, and HC levels between the control and LP treatments of the alfalfa and Chinese leymus silages. For the alfalfa silage, the CP content of the LP treat ment was significantly (p<0.05) higher than that of the control. For the Chinese leymus silage, the ADF content of the LP treatment was significantly (p<0.01) lower than that of the control.

Vitamin concentrations of alfalfa and Chinese leymus silages
The vitamin concentrations in the alfalfa and Chinese leymus silages are listed in   ensiling, the thiamin, riboflavin, niacin and pantothenic acid content of LPtreated silage was significantly (p<0.05) lower than that of untreated silage for both alfalfa and Chinese ley mus silages. The αtocopherol content of LPtreated silage was significantly (p<0.05) higher than that of untreated silage for the alfalfa silage. There was no significant (p>0.05) difference in pyridoxine content between the control and LP treatments for the alfalfa and Chinese leymus silages.

DISCUSSION
By the end of ensiling, the addition of LP inoculant had a positive effect on alfalfa and Chinese leymus silage fermen tation, as demonstrated by the low pH and butyric acid and ammonia nitrogen content and high lactic acid concentra tion in the inoculated silage. After 45 days of ensiling, the loss of WSC content in both forages was approximately 80%, which was due to the conversion of WSC to organic acid, while there was no significant difference in residual WSC content between the control and LPtreated silages. The pH of the alfalfa silage was much higher than that of the Chinese leymus silage, pro bably due to the high buffering capacity and low WSC content of alfalfa. The butyric acid and ammonia nitrogen concen trations in the alfalfa silage were significantly higher than those in the Chinese leymus silage in the present study, which in dicates that alfalfa undergoes a greater degree of proteolysis than Chinese leymus. This result is consistent with previous observations of Papadopoulos and Mckersie [19] and can perhaps be explained by the high susceptibility of alfalfa pro teins to proteolysis [20]. The higher acetic acid content and lower ratio of lactic acid to acetic acid in the control alfalfa silage than in the control Chinese leymus silage were indica tive of the activity of heterofermentative lactic acid bacteria. Based on the Flieg's points of the alfalfa and Chinese leymus silages, the fermentation quality of the Chinese leymus silage was better than that of the alfalfa silage, and the use of LP inoculant could improve the fermentation quality of both silages.
Many studies have investigated the αtocopherol and β carotene present in silage [69], while the B vitamins in alfalfa and Chinese leymus silages have not yet been discussed in the literature [7]. The thiamine concentrations in the alfalfa and Chinese leymus silages were 2.475 and 1.663 mg/kg DM, respectively, which were close to the level (1.37 mg/kg DM) reported by Beaudet et al [21]. The riboflavin levels in the al falfa and Chinese leymus silages were 52.21 and 42.53 mg/kg DM, respectively, which were higher than the value reported by Schwab et al [22] in corn silage (3.5 mg/kg DM) but similar to the value reported by Beaudet et al [21] in corn silage (73.2 mg/kg DM). The niacin levels in the alfalfa and Chinese ley mus silages were 0.743 and 0.558 mg/kg DM, respectively, which were lower than value (1.1 to 34 mg/kg) previously reported by Ballet al [7] in silage. The pantothenic acid levels in the alfalfa and Chinese leymus silages were 18.38 and 14.89 mg/kg DM, respectively, which were higher than the value reported by Schwab et al [22] in corn silage (1.5 mg/kg DM). The pyridoxine levels in the alfalfa and Chinese leymus si lages were 1.140 and 1.108 mg/kg DM, respectively, which were lower than the value reported by Schwab et al [22] in corn silage (1.9 mg/kg DM). Based on the results of the pres ent study, the thiamin, riboflavin, niacin, pantothenic acid, pyridoxine and αtocopherol levels in the alfalfa forage were significantly higher than those in the Chinese leymus. The observed variability in B vitamin levels in silage suggests that many factors contribute to the variability, including forage species, climatic conditions, maturity and storage methods [7]. After 45 days of ensiling, the thiamin, riboflavin, niacin, pantothenic acid and pyridoxine concentrations in the un treated alfalfa silage decreased by 72.1%, 42.2%, 61.9%, 42.2%, and 57.6%, respectively. For the untreated Chinese leymus silage, the levels of thiamin, riboflavin, niacin, pantothenic acid and pyridoxine decreased by 54.1%, 60.2%, 64.8%, 60.3%, and 66.4%, respectively. Except for pyridoxine in Chinese ley mus, the loss of B vitamins increased upon inoculation with LP. The decrease in thiamine content in the present study was similar to the results of Rao and Basu [23], who found that thiamine levels decreased during fermentation with lactoba cilli. However, the reason underlying the observed influence of the lactic acid bacterial inoculant on B vitamin content in the present study. The continued increase in dairy productivity and quality of ruminants has resulted in increased B vitamin requirements [7]. Many studies conducted on other fermen tation products, such as yogurt, cheeses, tarhana, and cereals [10,2426], have shown that the levels of Bgroup vitamins in these fermented products increased after fermentation due to the synthesis of B vitamins by selected lactic acid bacteria [26]. Therefore, it may be possible to select Bvitaminproduc ing lactic acid bacteria to increase the B vitamin concentrations in silage.
αTocopherol is an important antioxidant. The αtocopherol content of the Chinese leymus forage was similar to the value reported by Ballet et al [7] in grasses harvested during the early to late flowering stages but higher than the value reported by Liu et al [9] for napier grass. After 45 days of fermentation, the αtocopherol content of the untreated Chinese leymus silage increased by 52.1% compared with that of raw Chinese leymus. To date, there has been no clear explanation for the changes in αtocopherol levels observed during ensiling. Lind qvist et al [27] hypothesized that αtocopherolproducing microorganisms exist on plants [28]. However, after 45 days of fermentation, the αtocopherol content of the untreated alfalfa silage decreased by 30.7% compared with that of raw alfalfa, while the αtocopherol content of the LPtreated alfalfa silage decreased by 8.0%; perhaps the αtocopherol content is associated with the pH of silage. Liu et al [9] found that the residual rate of αtocopherol was high at pH 4.0, and higher or lower pH values decreased the residual rate of α tocopherol. Therefore, the high pH of LPtreated and untreated alfalfa silage (4.82 and 5.22, respectively) resulted in loss of αtocopherol in alfalfa silage. The LPtreated silage retained a higher amount of αtocopherol than the control silage due to the low pH of the LPtreated silage.
In conclusion, prior to ensiling, the levels of five Bgroup vitamins (thiamin, riboflavin, niacin, pantothenic acid, and pyridoxine) and αtocopherol in alfalfa were significantly higher than those in Chinese leymus. With or without LP inoculation, the levels of the five Bgroup vitamins in alfalfa and Chinese leymus decreased after 45 days of ensiling, while the αtocopherol content of Chinese leymus increased. The LP inoculant improved the fermentation quality of both the alfalfa and Chinese leymus silages but increased the thiamin, riboflavin, niacin and pantothenic acid loss in the two forages after 45 days of fermentation. In addition, after fermentation, the αtocopherol content of alfalfa decreased, but the LPtreated silage retained more αtocopherol than the untreated silage.

CONFLICT OF INTEREST
We certify that there is no conflict of interest with any financial organization regarding the material discussed in the manu script.