Role of laccase and xylanase, with or without ferulic acid esterase-producing Lactiplantibacillus plantarum, on the aerobic stability, protease activity, microbial composition and in vitro degradability of mulberry silage

Role of laccase and xylanase, with or without ferulic acid esterase-producing Lactiplantibacillus plantarum, on the aerobic stability, protease activity, microbial composition and in vitro degradability of mulberry silage | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Role of laccase and xylanase, with or without ferulic acid esterase-producing Lactiplantibacillus plantarum, on the aerobic stability, protease activity, microbial composition and in vitro degradability of mulberry silage Ya Su, Qiang Yu, Yulong Xi, Yuanjiang Rong, Yixi Long, Yixiao Xie, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6626039/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 16 Jul, 2025 Read the published version in BMC Microbiology → Version 1 posted 19 You are reading this latest preprint version Abstract Laccase (L), xylanase (X), and ferulic acid esterase (FAE) act on lignin - carbohydrate complexes. Whether these enzymes, alone or combined, can improve mulberry ensiling and aerobic stability is unclear. This study assessed the effects of L, X, and FAE - producing Lactiplantibacillus plantarum (LP) on whole - plant mulberry silage's fermentation quality, aerobic stability, and microbial communities during aerobic exposure. After 60 days of ensiling, mulberry silage treated with distilled water (CK), LP, laccase + xylanase (LX), or LX + LP (M) was unsealed for 1, 3, 5, or 7 days for exposure to air. The results indicated that the LP and M treatments decreased mulberry silage pH. Lower aminopeptidase and carboxypeptidase activities likely reduced CP degradation and NH₃-N content (P < 0.05), while increasing LA and WSC production. Compared with the CK treatment, the addition of LX and M increased the AA content by 1.49-2.68-fold, indicating greater aerobic stability ( P < 0.05), which contributed to maintaining the storage quality of the silages during aerobic exposure. The application of additives to mulberry silage reduced the species richness; specifically, the additive treatments led to an increase in the relative abundance of Kondoa and Lentilactobacillus while decreasing that of Enterococcus and Delftia . Notably, Lentilactobacillus exhibited the capacity to inhibit the growth of other harmful microorganisms and emerged as the dominant genus within the LX group. In conclusion, treatment with the combination of laccase, xylanase, and FAE-producing L. plantarum can serve as an effective method to improve the silage quality and aerobic stability of mulberry. Mulberry ensiling Ferulic acid esterase Lactic acid bacteria Laccase and xylanase Aerobic stability Bacterial community Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Introduction China has long experienced a shortage of high - quality feed resources, especially high-quality protein feeds. This situation increases feed costs and reduces the efficiency of the livestock industry [ 1 ]. Therefore, developing new natural resources, such as nutrient - rich woody plants, is crucial for addressing the feed shortage resulting from increased animal husbandry. Mulberry ( Morus alba L.) is rich in protein, minerals, bioactive substances, and other nutrients and can be considered a new resource for replacing traditional ruminant feeds and alleviating the shortage of traditional protein raw materials [ 2 ]. Moreover, it can improve animal health and the quality of products. For example, it can increase the amino acid and fatty acid content in meat, enhancing the nutritional value and market competitiveness of meat products [ 3 ]. However, mulberry silage lacks lactic acid bacteria (LABs) and has a high buffering capacity and lignocellulose level, leading to subpar ensiling results [ 4 , 5 ]. Inoculation with LABs can improve fermentation, inhibit the growth of harmful microbes such as yeasts and moulds and increase the aerobic stability of silage [ 6 ]. The addition of cellulase and LABs can increase the quality of mulberry silage, conserve more nutrients and increase the antioxidant ability of the feed [ 7 ]. Using FAE-producing LABs in silage can promote lignocellulose decomposition and the release of free ferulic acid, enhancing silage quality and antioxidant properties [ 8 ]. Moreover, our team reported that the use of FAE-producing L. plantarum in combination with lignocellulose hydrolase significantly improved the fermentation quality of papyrus silage and increased the lignocellulosic content in Broussonetia papyrifera [ 9 ]. Lignocellulose consists of lignin, cellulose, and hemicellulose linked by various chemical bonds, forming a complex matrix [ 10 ]. Lignin and hemicellulose increase the difficulty of cellulose degradation by forming lignin‒carbohydrate complexes around the cellulose [ 11 ]. Xylan, a hemicellulose, is located in the secondary wall and is the second most abundant plant polysaccharide [ 12 ]. Xylanase can degrade xylan into monosaccharides and sever lignin‒carbohydrate bonds [ 13 ]. Laccase is a copper-containing phenol oxidase that can oxidize lignin, form high-potential free radicals, and degrade lignin by opening the aromatic ring, resulting in an increase in reducing-sugar recovery during saccharification [ 14 ]. In silage, laccases can create anaerobic conditions for LAB growth, reducing storage losses from plant cell respiration and aerobic microbe metabolism [ 15 ]. FAE can break lignin‒hemicellulose linkages, increase the accessibility of degrading enzymes to lignocellulose, and improve the lignocellulose degradation rate and the FAE product can break the linkages between lignin and structural polysaccharides, thereby increasing the in vitro degradability of lignocellulose [ 16 , 17 ]. The combined use of these three enzymes may increase the rate of degradation of the lignin-carbohydrate complex and promote the release of ferulic acid, which has strong antioxidant and antibacterial properties [ 18 ]. However, the combined use of these three enzymes in mulberry silage and their aerobic stability have not been reported. Therefore, whether the combination of FAE-producing L. plantarum with laccase and xylanase can have a positive effect on the aerobic stability of mulberry silage needs to be explored. This study aimed to examine the synergistic effects of laccase, xylanase, and FAE-producing L. plantarum on mulberry silage by exploring the aerobic stability, protease activity, microbial composition, in vitro degradability, fermentation characteristics, antioxidant activity and microbial communities during aerobic exposure, thereby providing a basis for the preservation of mulberry. Materials and methods Raw materials and additives The mulberry (second crop, tasseling stage) was harvested on 8 September 2023 at the experimental site in Huaxi District, Guiyang City, Guizhou Province. The FAE-producing L. plantarum used in this study were obtained from a previous isolation by our research team [ 18 ]. xylanase (X, 100 U mg − 1 ) was obtained from Shanghai Macklin Biochemical Technology Co., Ltd and laccase (L, 10 U mg − 1 ) was obtained from Beijing Xiasheng Biotechnology Development Co., Ltd. Preparation of silage and aerobic stability analysis Preparation of mulberry silage, the treatment groups were: Control group (CK) without the addition of bacteria and enzymes, Experimental group (LP) with the addition of FAE-producing strain L. plantarum at an addition of 1 × 10 6 CFU/g FW, the experimental group (LX) with the addition of 25 U/g + 25 U/g FW laccase and xylanase, and the experimental group ((M) with the addition of FAE-producing L. plantarum , laccase and xylanase, respectively, and set up 4 replicates for each treatment. The bags were filled with 400 g/bag and then vacuum sealed for silage (store at room temperature away from light), the bags were opened after 60 days of silage and samples were taken at 0, 1, 3, 5 or 7 days after aerobic exposure to analyze the nutritional quality, fermentation quality and aerobic stability of each treatment, as well as in vitro fermentation after 60 days of fermentation. After opening the bag, the feed is exposed to the air and covered with gauze to prevent contamination by other impurities such as fruit flies and moisture dissipation, and the gas can freely enter the silage bag. Multiple probes of the multi-channel temperature recorder are placed in the center of the feed, and three blanks are set at the same time, and the temperature is recorded at 30-minute intervals. If the sample temperature is higher than the ambient temperature by 2°C, it means that the silage feed is beginning to rot and deteriorate, and the time is recorded [ 20 ]. Fermentation parameters and chemical composition The fermented mulberry silage samples were opened and mixed well, 10 g of each sample was weighed and put into a self-sealing bag, then 90 mL of sterile water was added, and leaching was carried out at 4℃ for 24 h, finally it was filtered through 4 layers of gauze to obtain the leachate of silage samples. It was stored in the refrigerator at -20℃ and then used to determine the pH value, organic acid and ammoniacal nitrogen content. The pH was determined using a pH meter (PHSJ-5T, INESA Scientific Instrument Co., Ltd., Shanghai, China). Organic acids, such as lactic acid (LA), acetic acid (AA), propionic acid (PA) and butyric acid (BA) were measured by liquid chromatography (Vanquish Core, Thermon Fisher Scientific, US) (KC-811 column; oven temperature: 50°C; flow rate: 1 mL min − 1 ) as the method of Li [ 21 ]. Ammoniacal nitrogen (NH₃-N) was determined using the phenol- sodium hypochlorite colorimetric method [ 22 ]. The 200 g of silage samples were taken and dried in an oven at 65℃ until constant weight to determine the dry matter (DM) content [ 23 ]. The dried samples were pulverized and sealed for storage. Crude protein (CP) was determined by the Kjeldahl method (VAPODEST500, C. Gerhardt GmbH & Co. KG, Germany) [ 23 ]. Neutral detergent fiber (NDF), acid detergent fiber (ADF), and acid lignin (ADL) were determined by the paradigm washed fiber method (ANKOM DELTA , ANKOM Technology, Macedon, NY, USA) [ 24 ]. Water-soluble carbohydrates (WSC) were determined by the enthrone- sulfate colorimetric method [ 25 ]. Protein hydrolase activity Upon the opening of the bag, 10 g of mulberry was sampled from each bag and homogenized with 90 mL of pre - cooled 0.1 M sodium phosphate buffer (pH 6.0, containing 5 mM sodium thiosulfate) for 60 s. The resulting homogenates were filtered through four layers of cheesecloth and subsequently centrifuged at 8,000 × g for 10 min at 4°C. The supernatant samples were stored at − 80°C for subsequent analysis of protease activities. The activities of aminopeptidase, carboxypeptidase, and acid protein - degrading enzyme were determined in accordance with the method described by Li [ 26 ]. Analyses of microbial counts The bacterial composition was analyzed using the plate counting method by mixing 20 g of sample with 180 mL of distilled water, shaking thoroughly, and performing serial dilutions (10⁻¹ to 10⁻⁷). Lactic acid bacteria were enumerated using De Man, Rogosa, and Sharpe (MRS) agar plates (Land Bridge, Beijing, China), which were incubated under anaerobic conditions at 37°C for 48 h. Coliform bacteria were enumerated using Erythromycin Agar plates (Land Bridge, Beijing, China), which were incubated at 30°C for 48 h. Yeasts were enumerated using Bengal Red Agar plates (Land Bridge, Beijing, China), which were incubated at 30°C for 48 h. Microbial community analysis Another 10 g of sample was divided into sealed bags and stored at -80°C in a freezer for the identification of the microorganisms in the sample. To further elucidate the dynamic changes in microorganisms during silage fermentation, DNA was extracted from the samples using the standard cetyltrimethylammonium bromide (CTAB) method for sequencing analysis and strain identification. Polymerase chain reaction (PCR) amplification was conducted with specific primers, using Barcode and GC Buffer from New England Biolabs (Ipswich, MA, USA), as well as high-fidelity and high-efficiency enzymes. The bacterial 16S rRNA gene was amplified using the primers F (5'-CCTAYGGGRBGCASCAG-3') and R (5'-GGACTACNNGGGTATCTAAT-3'). The fungal ITS rRNA gene was amplified using the primers F (5'-CTTGGTCATTTAGAGGAAGTAA-3') and R (5'-GCTGCGTTCTTCATCGATGC-3'). The bacterial 16S amplification protocol was as follows: initial denaturation at 95°C for 2 minutes, followed by 25 cycles of denaturation at 95°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 30 s, followed by a final extension was performed at 72°C for 5 minutes. Small fragment libraries were constructed on the basis of the characteristics of the amplified regions; to determine the species composition of the samples, species annotation and abundance analysis of the validated data were performed by Novogene Technology Co., Ltd (Inner Mongolia, China). Subsequently, alpha diversity and coverage value analyses were conducted using the Magic platform ( https://magic.novogene.com/ ) to assess differences in community structure. In vitro rumen fermentation trial Rumen fluid was collected and filtered through four layers of gauze into preheated anaerobic thermos flasks and stored until use. In vitro digestion medium was prepared according to the methods of Zhou et al [ 27 ]. Mulberry feed samples were reweighed [1.00 g dry matter] into polyester bags, sealed, and placed in 250 ml fermentation flasks. Two hundred milliliters of the rumen fluid mixture was subsequently dispensed into the preheated flasks at 39°C, and the flasks were closed with plastic caps equipped with one-way valves to prevent the accumulation of fermentation gases after the flasks were filled with CO 2 . The flasks were then transferred to a shaker incubator and shaken at 125 r/min for 72 h at 39°C. After 72 h of incubation, the nylon bags were removed and rinsed with water. The bags were then dried at 65°C until a constant weight was reached. In vitro DM digestibility (IVDMD), NDF digestibility (IVNDFD), and ADF digestibility (IVADFD) were calculated from the change in weight after the reaction. Statistical Analysis Multifactorial analysis of variance (ANOVA) (one-way or two-way) was performed mainly on the fixed effects of additives and Aerobic exposure time using SPSS 21 software (IBM Corp., New York, NY, USA). Microbial data were normalized by log 10 -transformation on a fresh weight basis. Duncan's and Tukey’s multiple comparison was used to determine the statistical difference between the means. Differences were considered significant when P < 0.05. Results Chemical composition of raw material before ensiling Table 1 Chemical composition and protein hydrolase activity of ensiling materials. Parameter Mean pH 6.64 DM (g/kg FM) 410.94 WSC (g/kg DM) 111.97 CP (g/kg DM) 115.43 NDF (g/kg DM) 409.24 ADF (g/kg DM) 275.98 ADL (g/kg DM) 72.75 Aminopeptidase (units h − 1 g − 1 DM of forage) 17.32 Carboxypeptidase (µmol of free amino acid released h − 1 g − 1 DM of forage) 6.95 Acid proteinase (units h − 1 g − 1 DM of forage) 69.03 DM, dry matter; FM, fresh matter; WSC, water-soluble carbohydrates; CP, crude protein.; NDF, neutral detergent fibers; ADF, acid detergent fiber; ADL, acid detergent lignin. As shown in Table 1 , before silage, the pH value of mulberry was 6.64, and the DM content reached 410.94 g/kg fresh matter (FM). The CP content was 115.43 g/kg DM, the NDF content was 409.24 g/kg DM, the ADF content was 275.98 g/kg DM, and the ADL content was 72.75 g/kg DM. Before ensiling, the aminopeptidase activity in mulberry was 17.32 units h⁻¹ g⁻¹ DM of forage, the acidic protein hydrolase activity was 69.03 units h⁻¹ g⁻¹ DM of forage, and the carboxypeptidase activity was 6.95 µmol h⁻¹ g⁻¹ DM. The mulberry had a WSC content of 111.97 g/kg DM, which met the ensiling requirements. Fermentation of silage during aerobic exposure Table 2 Changes in fermentation during aerobic mulberry exposure. Parameter Treatment (T) Days of aerobic exposure (D) SEM P -value Day 0 Day 1 Day 3 Day 5 Day 7 T D T × D pH CK 4.71Ac 4.83Abc 4.94Abc 5.76Aab 6.09Aa 0.087 < 0.001 < 0.001 0.001 LP 4.20Bb 4.21Cb 4.26Cb 4.27Bb 4.97Ba LX 4.66Ac 4.68Bbc 4.73Bab 4.76Bab 4.74Ba M 4.17Bb 4.19Cb 4.21Cb 4.20Bb 5.53ABa LA (g/kg DM) CK 40.34Db 54.36Ba 29.36Dc 13.24Cd 7.12Ce 0.380 < 0.001 < 0.001 < 0.001 LP 69.75Bb 89.35Aa 56.00Bc 25.87Bd 22.92Ad LX 43.49Cb 59.05Ba 37.81Cc 23.41Bd 23.06Ad M 83.43Aa 91.84Aa 66.46Ab 34.65Ac 13.58Bd AA (g/kg DM) CK 2.09Dab 2.37Ca 1.71Cbc 0.90Dd 1.39Bcd 0.032 < 0.001 < 0.001 < 0.001 LP 2.49Cb 3.05Ba 2.32Bb 1.63Cc 3.06Aa LX 3.52Aa 3.63Aa 2.56Bc 2.85Abc 3.31Aab M 3.13Bb 3.67Aa 3.42Aab 2.41Bc 2.51Ac PA (g/kg DM) CK 0.48b 0.58a 0.59a ND ND 0.005 < 0.001 < 0.001 < 0.001 LP 0.27 0.27 ND ND ND LX 0.2 0.14 ND ND ND M ND ND ND ND 0.59 BA (g/kg DM) CK ND ND ND ND ND 0.013 < 0.001 < 0.001 < 0.001 LP ND 3.26Ba 3.50a ND ND LX ND ND ND ND ND M ND 4.32Aa 3.52b ND ND NH 3 –N (g/kg TN) CK 0.6Ab 0.6Ab 0.5ABb 0.9Bb 1.8Ba 0.02 < 0.001 < 0.001 < 0.001 LP 0.1Bb 0.2Cb 0.3Bb 0.2Cb 0.7Ca LX 0.4Ac 0.4Bc 0.7Abc 1.1Ab 2.4Aa M 0.1Bc 0.2BCbc 0.3Bb 0.2Cbc 0.7Ca LA, lactic acid; AA, acetic acid; PA, propionic acid; BA, butyric acid; NH 3 –N, ammoniacal nitrogen; TN: total nitrogen. CK, control; LP, FAE-producing L. plantarum ; LX, laccase and xylanase; M, combination of FAE-producing L. plantarum , laccase and xylanase. T, Treatment; D, Aerobic exposure days, T × D, Interacting Treatment and Days of Aerobic Exposure; SEM, standard error of the mean; A−D Within a column and item, means without a common superscript differed ( P < 0.05); a−e Within a line, means without a common superscript differed ( P < 0.05). The data in Table 2 show that the application of additives significantly reduced the pH of the forage mulberry silage ( P < 0.05 ). During the first 5 days of aerobic exposure, the pH values of the groups treated with FAE-producing L. plantarum (LP and M) were significantly lower than those of the groups without L. plantarum (CK and LX) ( P < 0.05 ). During aerobic exposure, the LA and AA contents in all the additive treatment groups were consistently significantly greater than those in the control group ( P < 0.05 ). The LA content increased by 6.46–44.09 g/kg DM, and the AA content increased by 0.46–1.13 g/kg DM. In particular, compared with the CK treatment, the laccase and xylanase treatments (LX and M) increased the AA content of mulberry silage by 1.49–2.68-fold. Additive-treated PA and BA levels were unsatisfactory, possibly due to temperature changes in the air during aerobic exposure. The NH 3 -N content increased with the increasing aerobic exposure time, the treatment groups containing FAE-producing L. plantarum (LP and M) was significantly lower than that of the treatment groups without the addition of FAE-producing L. plantarum (LX and CK) ( P < 0.05 ). The NH 3 -N levels were significantly lower in the LX treatment than in the CK treatment for the first 3 days of aerobic exposure ( P < 0.05 ). Impact of additives on the aerobic stability of mulberry According to the temperature pattern during aerobic exposure shown in Fig. 1 A, the temperature of the CK group exceeded the ambient temperature by 2°C from 69–71 h of aerobic exposure, which indicates that the forage began to deteriorate during this period. When the temperature increased to less than 2°C, the silage was relatively stable and less prone to spoilage. During aerobic exposure, the LX treatment group did not exhibit obvious signs of aerobic spoilage. The LP treatment group first exceeded the ambient temperature by 2°C from 132–146 h, whereas the M treatment group reached this threshold (2°C above the ambient temperature) between 144–146 h. This sustained temperature increase indicated the onset of mulberry forage decay. As shown in Fig. 1 B, the aerobic stabilization time of the additive-treated groups was significantly longer than that of the CK group ( P < 0.05), and the aerobic stability, in descending order, was as follows: LX, M, LP, and CK. Effect of additives on the chemical composition of mulberry silage As shown in Fig. 2 a, with increasing aerobic exposure time, the DM content of each treatment group gradually decreased. During the first 5 days of aerobic exposure, the DM content of the M treatment group was significantly greater than that of the other treatment groups ( P < 0.05 ). This could be attributed to the synergistic effect among laccase, xylanase, and FAE-producing L. plantarum . Figure 2 b and 2 c indicates that the crude protein content gradually decreased as the aerobic exposure time increased. There was no significant difference among the treatments during the first 5 days of aerobic exposure, whereas on the 7th day, the M treatment exhibited significantly greater effects than did the control ( P < 0.05 ). The additive treatments (LP, LX and M) increased the WSC content by 0.78–7.0 g.kg − 1 DM. The WSC content of the LP and M groups was significantly greater than that of the FAE-producing L. plantarum groups (LX and M) ( P < 0.05 ). The contents of NDF and ADF in the LP, LX and M treatments were significantly lower than those in the CK treatment ( P < 0.05 ) (Fig. 2 d, e, f), especially the LP treatment is the most effective. The additive treatments had significantly lower levels of ADL than controls on days 0 and 7 of aerobic exposure ( P 0.05 ). Effect of additives on the lignocellulose of mulberry silage There was no significant difference in the cellulose content of mulberry silage during the first 5 days of aerobic exposure, but the additive treatment was significantly lower than CK on the 7th day of aerobic exposure ( P < 0.05 ), especially the M treatment had the lowest content (Fig. 3 a). During aerobic exposure, hemicellulose content of additive treatments was significantly lower than that of CK ( P < 0.05 ). As shown in Fig. 3 c, and lignin content of additive treatment group was significantly lower than that of control group on days 3–7 of aerobic exposure ( P 0.05 ). Changes in the activities of three types of proteases during aerobic exposure of mulberry silage As depicted in Fig. 4 a, b, c, with increasing aerobic exposure time, the activities of the three proteases increased. During aerobic exposure, the aminopeptidase activities of FAE-producing L. plantarum (LP and M) treatments were significantly lower than those of CK and LX treatments ( P 0.05 ). Carboxypeptidase activities of all additive treatments were significantly lower than those of the CK treatment during the first 5 days of aerobic exposure ( P < 0.05 ) and were not significantly different on day 7. Acidic protein hydrolase activity was significantly higher in LP and M treatments than in CK and LX ( P 0.05 ). In vitro digestibility analysis of mulberry fermentation for 60 days Figure 5 shows that FAE-producing L. plantarum (LP and M) treatments increased IVDMD ( P 0.05 ), and no significant difference between LP and M ( P > 0.05 ). IVNDFD was significantly higher in the LX treatment than in M, which was significantly higher than in the CK and LP treatments ( P 0.05 ). Effects of additive treatments on IVADFD ( P > 0.05 ). Changes in microbial communities during aerobic exposure of mulberry silage As shown in Table 3 , as the duration of aerobic exposure increased, the LAB population gradually increased. Conversely, yeast and Coliform bacteria started to grow from an initial count of zero. Compared with those in the LX and control groups, significantly lower levels were detected in the LP and M treatments ( P < 0.05 ). In both the LP and M groups, the LAB population tended to increase with increasing pH. At the commencement of aerobic exposure (day 0), no growth of Coliform bacteria or yeast was detected in any of the treatments. In the CK treatment, the growth of Coliform bacteria and yeast commenced on day 1 of aerobic exposure. The additive treatment significantly reduced the number of yeasts ( P < 0.05 ). Table 3 Changes in microbial counts during aerobic exposure of silage mulberry Parameter Treatment (T) Days of aerobic exposure (D) SEM P-value Day 0 Day 1 Day 3 Day 5 Day 7 T D T × D LAB(lg CFU/gFM) CK 5.53Bc 5.64c 6.19Ab 6.48Ab 7.41Aa 0.021 < 0.001 < 0.001 < 0.001 LP 4.15Cd 4.98Cc 4.92Bc 6.15Bb 6.93Ba LX 6.09A 6.20A 6.07A 5.47C 5.63C M 4.00De 4.39Dd 5.04Bc 6.28ABb 6.98Ba Coliform bacteria (lg CFU/gFM) CK ND 5.72Ad 6.07Ac 6.32Ab 7.06Aa 0.013 < 0.001 < 0.001 < 0.001 LP ND 5.11Bd 4.74Bc 6.20Ab 6.84Ba LX ND ND ND 4.15Bb 5.00Ca M ND ND 4.73Bc 6.16Ab 6.99ABa Yeast (lg CFU/g FM) CK ND 6.03Ac 6.07Ac 6.46Ab 6.91Aa 0.011 < 0.001 < 0.001 < 0.001 LP ND ND ND 6.11Bb 6.76Ba LX ND 4.83B ND ND ND M ND ND 4.24Bc 6.18Bb ND LAB, lactic acid bacteria; CK, control; LP, FAE-producing L. plantarum ; LX, laccase and xylanase; M, combination of FAE-producing L. plantarum , laccase and xylanase. T, Treatment; D, Aerobic exposure days, T × D, Interacting Treatment and Days of Aerobic Exposure; SEM, standard error of the mean; A−D Within a column and item, means without a common superscript differed ( P < 0.05); a−e Within a line, means without a common superscript differed ( P < 0.05). As presented in Fig. 6 , Component 1 and Component 2 explained 69.29% and 10.33% of the total variance, respectively. After 60 days of ensiling, on the third day of aerobic exposure, the LP, LX, and M feed samples were clearly differentiated from CK. By the seventh day of aerobic exposure, LP and M were distinctly separated from LX and CK. This result demonstrated significant disparities in the microbial communities between inoculated and noninoculated silage. The microbial community structure depicted in Fig. 7 a indicates that, following ensiling, in aerobically exposed whole-plant mulberry silage, the bacterial community was dominated by the phyla Firmicutes and Proteobacteria . The LX treatment significantly increased the relative abundance of Firmicutes . Conversely, the M treatment led to a notable increase in the relative abundance of Proteobacteria on both day 0 and day 7. As shown in Fig. 7 b, the bacterial composition of mulberry silage is predominantly characterized by the genera Lactiplantibacillus , Lentilactobacillus , and Stenotrophomonas . The introduction of additives led to a reduction in the richness of bacterial species in mulberry silage. Lentilactobacillus represents the dominant microflora in LX silage. LP treatment resulted in a significant increase in the relative abundance of Lactiplantibacillus during the first 3 days of aerobic exposure and a decrease on day 7. As depicted in Fig. 8 , Ascomycetes was the dominant phylum throughout the aerobic phase of mulberry silage fermentation. The additive treatments induced a statistically significant reduction in the relative abundance of Issatchenkia during the aerobic exposure of mulberry. Notably, within the initial three - day period of aerobic exposure in the LX group, Issatchenkia was scarcely detectable. Treatment with FAE - producing L. plantarum (LP and M) increased the relative abundance of Kazachstania on days 3 and 7 of aerobic exposure. The additive treatments increased the relative abundance of Phyllactinia , especially in the LX treatment group on days 3 and 7 of aerobic exposure. The LX treatment also increased the relative abundance of Cladosporium in mulberry silage on days 3 and 7 of aerobic exposure. As depicted in Fig. 9 , the NH₃–N content of mulberry silage was negatively correlated with the relative abundance of Lactiplantibacillus within the bacterial community ( P < 0.05 ). The AA content was positively correlated with the relative abundances of Lacticaseibacillus and Lentilactobacillus ( P < 0.01 ) but negatively correlated with the relative abundance of Chryseobacterium ( P < 0.05 ). The WSC content was negatively correlated with the relative abundances of Enterobacter and Escherichia-Shigella ( P < 0.01 ). Conversely, the WSC content was positively correlated with the relative abundance of Delftia ( P < 0.01 ). The activity of acidic protein hydrolase was positively correlated ( P < 0.01 ) with the relative abundance of Stenotrophomonas , whereas the relative abundance of Chryseobacterium was negatively correlated ( P < 0.01 ) with that of Enterobacter . Discussion Compared with corn and oat, which are typical conventional forages, mulberry exhibits higher levels of CP, NDF, ADF, and ADL [ 28 , 29 ]. The high fiber content can restrict the efficiency of mulberry utilization; consequently, fiber degradation is crucial for enhancing the utilization of mulberry silage. In the LP treatment, the NDF and ADF contents were significantly lower, likely because the FAE-producing L. plantarum degraded hemicellulose [ 30 ], leading to a significantly reduced hemicellulose content in the LP treatment. At the later stage of aerobic exposure, the ADL content in the additive treatment was significantly lower than that in the CK group, which can be attributed to the certain delignification ability of the FAE-producing L. plantarum [ 31 ]. Cellulase and xylanase hydrolyze plant cell walls to generate fermentable sugars, thereby decreasing the NDF and ADF content in oat silage [ 12 ]. In this study, the combined treatment of laccase and xylanase had a similar effect. Xylanase breaks the chemical bond between lignin and carbohydrates, feruloyl esterase can cleave the ester bond between lignin and hemicellulose, and laccase degrades lignin. Xylanases hydrolyze hemicelluloses, thereby improving the accessibility of cellulases to cellulose [ 32 ]. When lignin is degraded, the strong bonds among lignin, cellulose, and hemicellulose are disrupted; as a result, the utilization of cellulose and hemicellulose is enhanced [ 33 ]. We hypothesized that during the ensiling process, the FAE-producing L. plantarum degraded lignocellulose [ 8 ]. The additives were more effective in fiber degradation during the later stages of aerobic exposure, thereby enhancing the effectiveness of mulberry silage during the later stages of aerobic exposure. Laccase and cellulase increase the IVDMD, while lactobacilli increase the IVNDFD. A higher IVDMD was associated with a lower NDF content, whereas the IVDMD was positively correlated with the CP content [ 34 ]. No substantial variation was detected in the IVADFD. Research has indicated that the molecular structure of carbohydrates can affect the rumen degradation rate of NDF in alfalfa and concentrate-based feed [ 35 ]. The chemical composition of the additive-treated sorghum silage, including its WSC and CP contents, may be optimal for rumen microbial degradation. This could also indirectly explain why the highest LA content and the lowest pH value were observed in the LP and M treatments. The disparities in IVNDFD between the control and additive treatments could be partly attributed to variations in NDF degradation rates [ 36 ]. An in-depth study revealed that FAE-producing bacteria play a key role in lowering the pH of silage. This finding was consistent with the results of previous research, indicating that these bacteria could effectively promote lactic acid production and acidification in silage, thus contributing to improving the preservation and quality of silage [ 37 ]. Aerobic spoilage of silage could be considered to have occurred when the pH of the silage increased by 0.5 from the initial value [ 38 ]. According to pH the mulberry silage control started to spoilage on aerobic day 5, LP and M started to spoilage on day 7, whereas LX remained essentially unaltered during aerobic exposure. The LA content significantly decreased after 1 day of aerobic exposure, which was attributed to the breakdown and utilization of LA by microorganisms such as yeasts and aerobes [ 39 ]. Additive treatments increased the AA content of mulberry silage during aerobic exposure. In silage fermentation, increasing the AA content could increase aerobic stability and maintain storage quality, inhibiting the growth of yeasts and undesirable bacteria and helping LAB become the dominant flora, thereby improving silage fermentation quality [ 40 ]. This is one of the reasons why the additive treatment improves aerobic stability. Aerobic microorganisms (yeast and aerobic bacteria) oxidize organic acids and WSCs to produce carbon dioxide and water, generating heat during growth and reproduction, which leads to an increase in temperature and results in a decrease in aerobic stability [ 41 , 42 ]. This was most likely because a large amount of AA was produced, which inhibited the growth of moulds and yeasts during aerobic exposure [ 43 ]. This is because the addition of FAE-producing LABs and degrading enzymes may lead to the generation and retention of more WSCs via the decomposition of lignocellulose and rapid and dominant lactic acid fermentation, which can inhibit the growth of undesirable bacteria during the aerobic stage [ 44 ]. The WSC content of the LX treatments was not significantly different from that of the control. This may be because the pH value of the silages did not decrease below 4.2, resulting in the continued utilization of sugar by other harmful microorganisms [ 45 ]. Carboxypeptidases, aminopeptidases and acid proteases are the main plant enzymes involved in protein degradation. These enzymes exhibit different behaviors under different pH and temperature conditions and have different sensitivities to inhibitors. The overall extent of protein hydrolysis in silage is largely dependent on pH, which has a strong influence on the activity and stability of proteases [ 8 , 26 ]. This suggests that the addition of FAE-producing L. plantarum facilitates the inhibition of protein degradation, thereby reducing the ammoniacal nitrogen content. This may be because the additive accelerates Lactobacillus fermentation and inhibits the activity of spoilage bacteria and the cellular respiration in harvested plant tissues [ 46 ].The higher ammoniacal nitrogen content of the LX treatments was probably due to the higher pH resulting in elevated protease activity and protein degradation. The increase in acid protease activity, in conjunction with an elevated abundance of peptides and free amino acids, strongly suggests the pivotal role of acid proteases in protein hydrolysis. Treatments involving FAE-producing L. plantarum (LP and M) increase acid protease activity by modulating the pH value. During aerobic exposure, the number of Lactiplantibacillus decreased, which may be due to the proliferation of Enterobacter during silage fermentation, which converted LA produced by Lactiplantibacillus to AA.Since AA is less acidic than LA, the increase in AA content will increase the pH value of the silage and disrupt the growth environment of Lactiplantibacillus [ 47 ].LX was the dominant bacterium during aerobic exposure, and several studies have shown that inoculation with Lentilactobacillus buchneri significantly improved the aerobic stability of corn silage. LX is the dominant bacterium during aerobic exposure, and the results of several studies have shown that inoculation with Lentilactobacillus buchneri significantly improves the aerobic stability of corn silage. This could also explain why the LX treatment had the best aerobic stabilization results. The Ascomycetes phylum was the dominant phylum throughout the aerobic phase of mulberry silage fermentation, which is in agreement with the previous findings of Liu et al [ 3 ]. Issatchenkia has been identified as the main microorganism responsible for aerobic deterioration of maize and barley silage [ 33 ]. Kazachstania unispora was the main yeast species observed in aerobically deteriorated maize silage one of them [ 48 ]. Once the silage is exposed to air, aerobic microorganisms use LA as a growth substrate, leading to an increase in the pH of the silage. A high-pH environment favours the growth of filamentous fungi, and the proliferation of filamentous fungi, in turn, causes an increase in silage temperature. This phenomenon explains why the aerobic stability of the additive-treated group was greater than that of the CK group. Lactobacillus stenotrophomonas was positively correlated with AA and has the ability to convert some of the LA to AA under acidic conditions as documented by Muck et al [ 49 ]. Lactobacillus lentilus increased AA content and thus improved aerobic stability. Stenotrophomonas was positively correlated with acidic proteases, Stenotrophomonas spp. have low nutrient requirements but are considered unfavorable for silage due to their ability to degrade proteins [ 50 ].The relative abundance of Lactiplantibacillus, Lentilactobacillus , Phyllactinia , Cladosporium and Kondoa was positively correlated (P < 0.05) with AA levels, which may be due to the fact that these taxa have different metabolic functions. In contrast, the relative abundance of Wickhamia was negatively correlated (P < 0.05) with AA levels. These findings suggest that these microorganisms may be involved in the aerobic stabilization of mulberry silage. Overall, the application of additives significantly enhanced the fermentation quality of mulberry silage. The M treatment exhibited a pronounced positive impact on in vitro fermentation. Moreover, these treatments effectively improved the aerobic stability of mulberry silage, with the LX treatment affording the highest degree of stability. The utilization of mulberry as feed not only broadens the spectrum of available feed resources but also enhances feeding efficiency. Conclusion This study revealed that the addition of FAE-producing L. plantarum , laccase and xylanase increased the LA and AA contents and decreased the pH of mulberry silage, reducing the proliferation of harmful bacterial strains. The aerobic stability of the LX treatment group was the greatest, and that of the M group was similar to that of the control group. Moreover, the additive treatments reduced the species richness of mulberry silage. Lentilactobacillus became the dominant bacterial genus in the LX treatment group, which might have contributed to the increase in the aerobic stability of the mulberry silage. Overall, the addition of laccase, xylanase and FAE L. plantarum synergistically improved the fermentation and aerobic stability of mulberry silage. Declarations Funding This work was supported by the National Key Research and Development Program of China (2022YFD1300900), Qian Ke He Ping Tai Ren Cai-BQW[2024]003，Qian Ke He Cheng Guo ([2022] Zhong Dian 005), and GZMARS-Forage Industry Technology System of Guizhou Province. Availability of data and materials The raw sequence data have been deposited in the sequence read archive at the NCBI (https://www.ncbi.nlm.nih.gov/) under accession number PRJNA1244789 Ethics approval and consent to participate Not applicable. Competing interests The authors declare no competing interests. References El Naggar S, El-Mesery H S. Azolla pinnata as unconventional feeds for ruminant feeding. Bulletin of the National Research Centre , 2022, 46 (1), 66. https://doi.org/10.1186/s42269-022-00752-w Song F, Li Y, Huang J, Lu W, Guo Z, Deng W. Integrated transcriptomic and proteomic analyses reveal the impact of drought and heat stress combination on Morus alba[J]. Environmental and Experimental Botany, 2024, 228(PA):105988-105988. https://doi.org/ 10.1016/j.envexpbot.2024.105988 Liu Y, Xiao Y, Xie J, Peng Y, Li F, Chen C, Yin Y, et al. Dietary supplementation with flavonoids from mulberry leaves improves growth performance and meat quality, and alters lipid metabolism of skeletal muscle in a Chinese hybrid pig. Animal Feed Science and Technology , 2022, 285, 115211. https://doi.org/10.1016/j.anifeedsci.2022.115211 Guo Y, Huang R, Niu Y, Zhang P, Li Y, Zhang W. Chemical characteristics, antioxidant capacity, bacterial community, and metabolite composition of mulberry silage ensiling with lactic acid bacteria. Frontiers in Microbiology, 2024, 15, 1363256. https://doi.org/10.3389/fmicb.2024.1363256 Yu Q, Su Y, Xi Y, Rong Y, Long Y, Xie Y, Zheng Y, et al. Comparison of the impacts of cellulase and laccase on fermentation quality, bacterial composition and in vitro degradability of anaerobic cofermentation derived from Sudan grass with mulberry under Lactobacillus plantarum and different lignocellulolytic enzyme inoculation. Chemical and Biological Technologies in Agriculture , 2025, 12(1), 41.https://doi.org/10.1186/s40538-025-00760-8 Wang T, Teng K, Cao Y, Shi W, Xuan Z, Zhou J, Zhong J, et al. Effects of Lactobacillus hilgardii 60TS-2, with or without homofermentative Lactobacillus plantarum B90, on the aerobic stability, fermentation quality and microbial community dynamics in sugarcane top silage. Bioresource Technology , 2020, 312, 123600. https://doi.org/10.1016/j.biortech.2020.123600 He Li Wen, Zhou Wei, Wang Cheng, Yang FuYu, Chen XiaoYang, Zhang Qing. Effect of cellulase and Lactobacillus casei on ensiling characteristics, chemical composition, antioxidant activity, and digestibility of mulberry leaf silage. 2019. https://doi.org/10.3168/jds.2019-16468 Li F H, Ding Z T, Chen X Z, Zhang Y X, Ke W C, Zhang X, Guo X S, et al. The effects of Lactobacillus plantarum with feruloyl esterase-producing ability or high antioxidant activity on the fermentation, chemical composition, and antioxidant status of alfalfa silage. Animal Feed Science and Technology , 2021, 273, 114835. https://doi.org/ 10.1016/j.anifeedsci.2021.114835 Yu Q, Xu J, Li M, Xi Y, Sun H, Xie Y, Zheng Y, et al. Synergistic effects of ferulic acid esterase‐producing lactic acid bacteria, cellulase and xylanase on the fermentation characteristics, fibre and nitrogen components and microbial community structure of Broussonetia papyrifera during ensiling. Journal of the Science of Food and Agriculture, 2024, 104(6), 3543-3558. https://doi.org/10.1002/jsfa.13239 Qu J, Tian Z, Zhang F, Si C, Ji X. Effect of ultrasound-assisted xylanase pretreatment on the soluble substances of poplar wood and its model construction. Advanced Composites and Hybrid Materials , 2024, 7(3), 77. https://doi.org/ 10.1007/s42114-024-00871-0 Zhang Y C, Wang X K, Lin Y L, Zheng Y L, Ni K K, Yang F Y. Effects of microbial inoculants on fermentation quality and aerobic stability of paper mulberry silages prepared with molasses or Cellulase. Fermentation, 2022, 8(4), 167. https://doi.org/10.3390/fermentation8040167 Liu W, Si Q, Sun L, Wang Z, Liu M, Du S, Jia Y, et al. Effects of cellulase and xylanase addition on fermentation quality, aerobic stability, and bacteria composition of low water-soluble carbohydrates oat silage. Fermentation, 2023, 9(7), 638. https://doi.org/ 10.3390/fermentation9070638 Xu Q, Zhong H, Zhou J, Wu Y, Ma Z, Yang L, Li X, et al. Lignin degradation by water buffalo. Tropical animal health and production, 2021, 53(3), 344. https://doi.org/10.1007/s11250-021-02787-z Zhang S, Dong Z, Shi J, Yang C, Fang Y, Chen G, Tian C, et al. Enzymatic hydrolysis of corn stover lignin by laccase, lignin peroxidase, and manganese peroxidase. Bioresource Technology , 2022, 361, 127699. https://doi.org/ 10.1016/j.biortech.2022.127699 Bao X, Feng H, Guo G, Huo W, Li Q, Xu Q, Chen, L, et al. Effects of laccase and lactic acid bacteria on the fermentation quality, nutrient composition, enzymatic hydrolysis, and bacterial community of alfalfa silage. Frontiers in Microbiology , 2022, 13, 1035942. https://doi.org/10.3389/fmicb.2022.1035942 Jin L, Duniere L, Lynch J P, McAllister T A, Baah J, Wang Y. Impact of ferulic acid esterase producing lactobacilli and fibrolytic enzymes on conservation characteristics, aerobic stability and fiber degradability of barley silage. Animal Feed Science and Technology , 2015, 207, 62-74. https://doi.org/ 10.1016/j.anifeedsci.2015.06.011 Krueger N A, Adesogan A T, Staples C R, Krueger W K, Dean D B, Littell R C. The potential to increase digestibility of tropical grasses with a fungal, ferulic acid esterase enzyme preparation. Animal Feed Science and Technology, 2008, 145(1-4), 95-108. https://doi.org/ 10.1016/j.anifeedsci.2007.05.042 Park C H, Yeo H J, Baskar T B, Park Y E, Park J S, Lee S Y, Park S U. In vitro antioxidant and antimicrobial properties of flower, leaf, and stem extracts of Korean mint. Antioxidants, 2019, 8(3), 75. https://doi.org/10.3390/ antiox8030075 Yu Q, Li M, Zhang Y, Xu J, Li P, Sun H, Chen C, et al. Effects of different cutting stages and additives on the fermentation quality and microbial Community of Sudangrass (Sorghum sudanense Stapf.) silages. Fermentation, 2023, 9(8), 777. https://doi.org/10.3390/fermentation9080777 Ranjit N K, Kung Jr L. The effect of Lactobacillus buchneri, Lactobacillus plantarum, or a chemical preservative on the fermentation and aerobic stability of corn silage[J]. Journal of Dairy Science, 2000, 83(3): 526-535. https://doi.org/10.3168/jds.S0022-0302(00)74912-5 Li F, Ding Z, Ke W, Xu D, Zhang P, Bai J, Guo X, et al. Ferulic acid esterase-producing lactic acid bacteria and cellulase pretreatments of corn stalk silage at two different temperatures: Ensiling characteristics, carbohydrates composition and enzymatic saccharification. Bioresource Technology , 2019, 282, 211-221. https://doi.org/10.1016/j.biortech.2019.03.022 Kleinschmit D H, Schmidt R J, Kung Jr L. The effects of various antifungal additives on the fermentation and aerobic stability of corn silage. Journal of dairy science , 2005, 88(6), 2130-2139. https://doi.org/ 10.3168/jds.S0022-0302(05)72889-7 AOAC. Official methods of analysis, 15th ed. AOAC, Arlington, VA.[J]. 1990. Van Soest P V, Robertson J B, Lewis B A. Methods for dietary fiber, neutral detergent fiber, and nonstarch polysaccharides in relation to animal nutrition. Journal of dairy science , 1991, 74(10), 3583-3597. https://doi.org/10.3168/jds.S0022-0302(91)78551-2 Owens V N, Albrecht K A, Muck R E, Duke S H. Protein degradation and fermentation characteristics of red clover and alfalfa silage harvested with varying levels of total nonstructural carbohydrates. Crop Science , ,1999, 39(6), 1873-1880. https://doi.org/10.2135/cropsci1999.3961873x Li X, Tian J, Zhang Q, et al. Effects of mixing red clover with alfalfa at different ratios on dynamics of proteolysis and protease activities during ensiling[J]. Journal of Dairy Science, 2018, 101(10): 8954-8964. https://doi.org/10.3168/jds.2018-14763 Zhou Y Q, Cheng Y F, Zhu W Y. The Process and Matters Needing Attention of in vitro Fermentation of Rumen Microorganisms, 2021, Bio-101 e2003663. https://doi.org/10.21769/BioProtoc.2003663 Celis-Alvarez M D, López-González F, Martínez-García C G, Estrada-Flores J G, Arriaga-Jordán, C. M. Oat and ryegrass silage for small-scale dairy systems in the highlands of central Mexico. Tropical Animal Health and Production, 2016, 48, 1129-1134. https://doi.org/10.3168/jds.2007-0358 Ferrero F, Tabacco E, Borreani G. Lentilactobacillus hilgardii inoculum, dry matter contents at harvest and length of conservation affect fermentation characteristics and aerobic stability of corn silage. Frontiers in Microbiology , 2021, 12, 675563. https://doi.org/10.3389/fmicb.2021.675563 Zhao J, Dong Z, Li J, Chen L, Bai Y, Jia Y, Shao T. Ensiling as pretreatment of rice straw: The effect of hemicellulase and Lactobacillus plantarum on hemicellulose degradation and cellulose conversion. Bioresource technology , 2018, 266, 158-165. https://doi.org/ 10.1016/j.biortech.2018.06.058 Li M, Zhang L, Zhang Q, Zi X, Lv R, Tang J, Zhou H. Impacts of citric acid and malic acid on fermentation quality and bacterial community of cassava foliage silage. Frontiers in Microbiology , 2020, 11, 595622. https://doi.org/10.3389/fmicb.2020.595622 Öhgren K, Bura R, Saddler J, Zacchi G. Effect of hemicellulose and lignin removal on enzymatic hydrolysis of steam pretreated corn stover. Bioresource technology , 2007, 98(13), 2503-2510. https://doi.org/10.1016/j.biortech.2006.09.003 Xu T, Du H, Liu H, Liu W, Zhang X, Si C, Liu P, Zhang K. Advanced nanocellulose-based composites for flexible functional energy storage devices. Adv Mater, 2021, 33:2101368. https://doi.org/ 10.1002/adma.202101368 Lema M, Felix A, Salako S, Bishnoi U. Nutrient content and in vitro dry matter digestibility of silages made from various grain sorghum and sweet sorghum cultivars. Journal of Sustainable Agriculture, 2000, 17(1), 55-70. https://doi.org/10.1080/09712119.2001.9706717 Zhu J, Li F, Wang Z, Shi H, Wang X, Huang Y, Li S. Effect of Anaerobic Calcium Oxide Alkalization on the Carbohydrate Molecular Structures, Chemical Profiles, and Ruminal Degradability of Rape Straw. Animals, 2023, 13(15), 2421. https://doi.org/10.3390/ani13152421 Wang S, Li J, Dong Z, Chen L, Shao T. Inclusion of alfalfa improves nutritive value and in vitro digestibility of various straw–grass mixed silages in Tibet. Grass and Forage Science, 2018, 73(3), 694-704. https://doi.org/10.1111/gfs.12365 Heinritz S N, Martens S D, Avila P, Hoedtke S. The effect of inoculant and sucrose addition on the silage quality of tropical forage legumes with varying ensilability. Animal Feed Science and Technology , 2012, 174(3-4), 201-210. https://doi.org/ 10.1016/j.anifeedsci.2012.03.017 Liu Q, Zhang J, Shi S, Sun Q. The effects of wilting and storage temperatures on the fermentation quality and aerobic stability of stylo silage. Animal Science Journal, 2011, 82(4), 549-553. https://doi.org/10.1111/j.1740-0929.2011.00873.x Liu Y, Li Y, Lu Q, Sun L, Du S, Liu T, Jia Y, et al. Effects of lactic acid bacteria additives on the quality, volatile chemicals and microbial community of leymus chinensis silage during aerobic exposure. Frontiers in Microbiology, 2022, 13, 938153. https://doi.org/ 10.3389/fmicb.2022.938153 Wu B, Ai J, Li T, Qin W, Hu Z, Siqin T, Niu H, et al. Fermentation quality, aerobic stability, and microbiome structure and function of Caragana korshinskii silage inoculated with/without Lactobacillus rhamnosus or Lactobacillus buchneri. Frontiers in Sustainable Food Systems, 2023, 7, 1255936. https://doi.org/10.3389/fsufs.2023.1255936 Mugabe W, Shao T, Li J, Dong Z, Yuan X. Effect of hexanoic acid, Lactobacillus plantarum and their combination on the aerobic stability of napier grass silage. Journal of Applied Microbiology , 2020, 129(4), 823-831. https://doi.org/ 10.1111/jam.14650 Bing W, Hailing L. Effects of mulberry leaf silage on antioxidant and immunomodulatory activity and rumen bacterial community of lambs[J]. BMC Microbiology,2021,21(1):250-250. https://doi.org/ 10.1186/s12866-021-02311-1 Wang Y, He L, Xing Y, Zheng Y, Zhou W, Pian R, et al. Dynamics of bacterial community and fermentation quality during ensiling of wilted and unwilted moringa oleifera leaf silage with or without lactic acid bacterial inoculants. mSphere , 2019, 4(4), e00341-19. https://doi.org/ 10.1128/mSphere.00341-19 Guo G, Yuan X, Li L, Wen A, Shao T. Effects of fibrolytic enzymes, molasses and lactic acid bacteria on fermentation quality of mixed silage of corn and hulless–barely straw in the T ibetan Plateau. Grassland Science , 2014, 60 (4), 240-246. https://doi.org/10.1111/grs.12060 Liu M, Sun L, Wang Z, Ge G, Jia Y, Du S. Effects of alfalfa hay to oat hay ratios on chemical composition, fermentation characteristics, and fungal communities during aerobic exposure of fermented total mixed ration. Fermentation , 2023, 9 (5), 480. https://doi.org/ 10.3390/fermentation9050480 Zhao J, Dong Z, Li J, Chen L, Bai Y, Jia Y, Shao T. Ensiling as pretreatment of rice straw: The effect of hemicellulase and Lactobacillus plantarum on hemicellulose degradation and cellulose conversion. Bioresource technology , 2018, 266, 158-165. https://doi.org/ 10.1016/j.biortech.2018.06.058 Peng S, Xie L, Cheng Y, Wang Q, Feng L, Li Y, Sun Y, et al. Effect of Lactiplantibacillus and sea buckthorn pomace on the fermentation quality and microbial community of paper mulberry silage. Frontiers in Plant Science , 2024, 15, 1412759. https://doi.org/10.3389/fpls.2024.1412759 Lu Q, Wang Z, Sa D, Hou M, Ge G, Wang Z, Jia Y. The potential effects on microbiota and silage fermentation of alfalfa under salt stress. Frontiers in microbiology, 2021, 12, 688695. https://doi.org/10.3389/fmicb.2021.688695 Muck R E, Nadeau E M G, McAllister T A, Contreras-Govea F E, Santos M C, Kung Jr L. Silage review: Recent advances and future uses of silage additives. Journal of dairy science , 2018, 101(5), 3980-4000. https://doi.org/10.3168/jds.2017-13839 Mu L, Wang Q, Wang Y, Zhang Z. Effects of cellulase and xylanase on fermentative profile, bacterial diversity, and in vitro degradation of mixed silage of agro-residue and alfalfa. Chemical and Biological Technologies in Agriculture, 2023, 10(1), 40. https://doi.org/10.1186/s40538-023-00409-4 Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6626039","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":457466233,"identity":"27326bd2-e37f-4427-b690-f9fe49706106","order_by":0,"name":"Ya Su","email":"","orcid":"","institution":"Guizhou University","correspondingAuthor":false,"prefix":"","firstName":"Ya","middleName":"","lastName":"Su","suffix":""},{"id":457466234,"identity":"6420a0a9-5482-4b2e-b1dc-03f41a8d71a9","order_by":1,"name":"Qiang Yu","email":"","orcid":"","institution":"Guizhou University","correspondingAuthor":false,"prefix":"","firstName":"Qiang","middleName":"","lastName":"Yu","suffix":""},{"id":457466235,"identity":"4af14cf6-8cbb-4e6f-b140-9bbf922623ff","order_by":2,"name":"Yulong Xi","email":"","orcid":"","institution":"Guizhou University","correspondingAuthor":false,"prefix":"","firstName":"Yulong","middleName":"","lastName":"Xi","suffix":""},{"id":457466236,"identity":"2773de02-3b3f-4dc4-a301-88161cd61d66","order_by":3,"name":"Yuanjiang Rong","email":"","orcid":"","institution":"Guizhou University","correspondingAuthor":false,"prefix":"","firstName":"Yuanjiang","middleName":"","lastName":"Rong","suffix":""},{"id":457466237,"identity":"296fdb9d-c17b-41bd-a73c-029ed29748fe","order_by":4,"name":"Yixi Long","email":"","orcid":"","institution":"Guizhou University","correspondingAuthor":false,"prefix":"","firstName":"Yixi","middleName":"","lastName":"Long","suffix":""},{"id":457466238,"identity":"401596cc-f4f2-4089-97d7-cce03075d326","order_by":5,"name":"Yixiao Xie","email":"","orcid":"","institution":"Guizhou University","correspondingAuthor":false,"prefix":"","firstName":"Yixiao","middleName":"","lastName":"Xie","suffix":""},{"id":457466240,"identity":"15e8ba54-5222-4c1d-b5e5-5f7392e0f00c","order_by":6,"name":"Hong Sun","email":"","orcid":"","institution":"Guizhou University","correspondingAuthor":false,"prefix":"","firstName":"Hong","middleName":"","lastName":"Sun","suffix":""},{"id":457466242,"identity":"d86a67e2-9b9e-453a-8dc6-6b226143e0f7","order_by":7,"name":"Jun Hao","email":"","orcid":"","institution":"Guizhou University","correspondingAuthor":false,"prefix":"","firstName":"Jun","middleName":"","lastName":"Hao","suffix":""},{"id":457466247,"identity":"92f595d9-6239-48dc-9e6e-bdf53fa1fca7","order_by":8,"name":"Fuyu Yang","email":"","orcid":"","institution":"Guizhou University","correspondingAuthor":false,"prefix":"","firstName":"Fuyu","middleName":"","lastName":"Yang","suffix":""},{"id":457466248,"identity":"de74badf-9007-4653-ab92-1b88a32e12a8","order_by":9,"name":"Yulong Zheng","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA5UlEQVRIiWNgGAWjYDACdgY2EGXHD+EyE6GFGaIlWbKBVC2MGw4Qq8XgMPuzBx931DIbn1/+TIKhwjqxgf3sAQJaGNINZ545zmd240GaBMOZ9MQGnrwEQlqOSfO2HWM2u3HgmARj2+HEBgkeAwJaGNuk/7YdY9w842CbBOM/orQws0kzttUwbuBvZpNgbCBCi+RhNjbJ3rYDyRI32JgtEo6lG7fx5ODXwne8/ZnEz7Y6O/7+4w9vfKixlu1nP4Nfi8IBMHWYgUEigYEBiCDRhA/IN4CpOgYG/gOE1I6CUTAKRsFIBQDPr0VVgGkklwAAAABJRU5ErkJggg==","orcid":"","institution":"Guizhou University","correspondingAuthor":true,"prefix":"","firstName":"Yulong","middleName":"","lastName":"Zheng","suffix":""}],"badges":[],"createdAt":"2025-05-09 07:23:23","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6626039/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6626039/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12866-025-04165-3","type":"published","date":"2025-07-16T15:57:19+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":83025365,"identity":"33a3bc3f-7db7-4b6b-af0a-8f97ef6003b2","added_by":"auto","created_at":"2025-05-19 08:21:41","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":195891,"visible":true,"origin":"","legend":"\u003cp\u003eDuring aerobic exposure: (A) temperature change; (B) aerobic stabilization time; AT+2°C, Ambient temperature plus 2°C; CK, control; LP, FAE-producing\u003cem\u003e L. plantarum\u003c/em\u003e; LX, laccase and xylanase; M, combination of FAE-producing \u003cem\u003eL. plantarum\u003c/em\u003e, laccase and xylanase; a-d indicate differences between treatments (\u003cem\u003eP\u0026lt;0.05\u003c/em\u003e).\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6626039/v1/948bf654403a380da7e55d07.jpeg"},{"id":83025366,"identity":"12607481-9448-4483-9953-531831b34ec9","added_by":"auto","created_at":"2025-05-19 08:21:41","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":427656,"visible":true,"origin":"","legend":"\u003cp\u003eChanges in (a) dry matter; (b) water-soluble carbohydrates; (c) crude protein; (d) neutral detergent fiber; (e) acid detergent fiber; and (f) acid detergent lignin during aerobic exposure of mulberry silage. CK, control; LP, FAE-producing \u003cem\u003eL. plantarum\u003c/em\u003e; LX, laccase and xylanase; M, combination of FAE-producing \u003cem\u003eL. plantarum\u003c/em\u003e, laccase and xylanase.D, Aerobic exposure days; a-d, indicate differences between treatments (\u003cem\u003eP \u0026lt;\u003c/em\u003e 0.05).\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6626039/v1/fc661d84861390633dfbb2b4.jpeg"},{"id":83026457,"identity":"e74c60da-55e2-4999-934e-df53489abb8c","added_by":"auto","created_at":"2025-05-19 08:29:41","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":284826,"visible":true,"origin":"","legend":"\u003cp\u003eChanges in (a) cellulose, (b) hemicellulose, and (c) lignin content of silage mulberry during the aerobic period under different treatments. CK, control; LP, FAE-producing \u003cem\u003eL. plantarum\u003c/em\u003e; LX, laccase and xylanase; M, combination of FAE-producing \u003cem\u003eL. plantarum\u003c/em\u003e, laccase and xylanase; T, Treatment; D, Aerobic exposure days, T × D, Interacting Treatment and Days of Aerobic Exposure; A-D, indicate differences between treatments (\u003cem\u003eP \u0026lt; \u003c/em\u003e0.05)\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6626039/v1/e0ee99347d129c015c0d1619.jpeg"},{"id":83025367,"identity":"ac085a2e-0b80-4877-87cb-196e4ceb5ef8","added_by":"auto","created_at":"2025-05-19 08:21:41","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":218006,"visible":true,"origin":"","legend":"\u003cp\u003eChanges in the activities of (a) aminopeptidase, (b) carboxypeptidases, and (c) acidic protein hydrolase during aerobic exposure of mulberry silage.CK, control; LP, FAE-producing \u003cem\u003eL. plantarum\u003c/em\u003e; LX, laccase and xylanase; M, combination of FAE-producing \u003cem\u003eL. plantarum\u003c/em\u003e, laccase and xylanase; T, Treatment; D, Aerobic exposure days, T × D, Interacting Treatment and Days of Aerobic Exposure; a-d, indicate differences between treatments (\u003cem\u003eP \u0026lt; 0.05\u003c/em\u003e).\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6626039/v1/80a1d3a3aba2700799c35a55.jpeg"},{"id":83025373,"identity":"8a5b0ce3-a849-4e2c-b3c2-66eade65023f","added_by":"auto","created_at":"2025-05-19 08:21:41","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":12828,"visible":true,"origin":"","legend":"\u003cp\u003eIn vitro digestibility of DM, NDF and ADF of mulberry silage for 60 days. IVDMD, in - vitro dry matter digestibility; IVNDFD, in - vitro neutral detergent fiber digestibility; IVADFD, in - vitro acid detergent fiber digestibility; CK, control; LP, FAE-producing \u003cem\u003eL. plantarum\u003c/em\u003e; LX, laccase and xylanase; M, combination of FAE-producing \u003cem\u003eL. plantarum\u003c/em\u003e, laccase and xylanase; A-D, indicate differences between treatments (\u003cem\u003eP \u0026lt;\u003c/em\u003e0.05).\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-6626039/v1/9829a526b188e1dd1e358738.png"},{"id":83025372,"identity":"a53f860e-4db1-4e5b-9a6f-9c48bfe87835","added_by":"auto","created_at":"2025-05-19 08:21:41","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":41390,"visible":true,"origin":"","legend":"\u003cp\u003ePrincipal coordinate analysis (PCoA) based on 0, 3 and 7 days of mulberry silage aerobic exposure. FM, fresh mulberry; CK, control; LP, FAE-producing \u003cem\u003eL. plantarum\u003c/em\u003e; LX, laccase and xylanase; M, combination of FAE-producing \u003cem\u003eL. plantarum\u003c/em\u003e, laccase and xylanase.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-6626039/v1/78e9bd7bd2e4d5dbf8b7b67a.png"},{"id":83025374,"identity":"4c3539a0-cee3-4733-ac16-76f66d541fc8","added_by":"auto","created_at":"2025-05-19 08:21:41","extension":"jpeg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":378006,"visible":true,"origin":"","legend":"\u003cp\u003eThe relative abundance of the bacterial community in mulberry silage on days 0, 3, and 7 of aerobic exposure, at (A) the phylum level and (B) the genus level. based FM, fresh mulberry; CK, control; LP, FAE-producing \u003cem\u003eL. plantarum\u003c/em\u003e; LX, laccase and xylanase; M, combination of FAE-producing \u003cem\u003eL. plantarum\u003c/em\u003e, laccase and xylanase.\u003c/p\u003e","description":"","filename":"floatimage7.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6626039/v1/38239c6f2bd9aea628911062.jpeg"},{"id":83026947,"identity":"62ac7c01-853f-4916-984b-35e5d5d9126f","added_by":"auto","created_at":"2025-05-19 08:37:41","extension":"jpeg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":351638,"visible":true,"origin":"","legend":"\u003cp\u003eThe relative abundance of the fungi community in mulberry silage on days 0, 3, and 7 of aerobic exposure, at (A) the phylum level and (B) the genus level.based FM, fresh mulberry; CK, control; LP, FAE-producing \u003cem\u003eL. plantarum\u003c/em\u003e; LX, laccase and xylanase; M, combination of FAE-producing \u003cem\u003eL. plantarum\u003c/em\u003e, laccase and xylanase.\u003c/p\u003e","description":"","filename":"floatimage8.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6626039/v1/a24e16f4158c882dbe74447b.jpeg"},{"id":83026946,"identity":"56bd8f8b-53b3-42b6-bedc-1e0f6c9c5436","added_by":"auto","created_at":"2025-05-19 08:37:41","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":151949,"visible":true,"origin":"","legend":"\u003cp\u003eSpearman analysis between silage parameters and bacterial genus (A) fungal genus (B) during aerobic exposure.LA, lactic acid; AA, acetic acid; DM, dry matter; WSC, water-soluble carbohydrates; NDF, neutral detergent fiber; ADF, acid detergent fiber; ADL, acid detergent lignin; CP, crude protein; NH\u003csub\u003e3\u003c/sub\u003e-N, ammonia nitrogen; *Significance at P \u003cem\u003e\u0026lt; 0.05\u003c/em\u003e; **significance at \u003cem\u003eP \u0026lt; 0.01\u003c/em\u003e; ***significance at P \u003cem\u003e\u0026lt; 0.001\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"floatimage9.png","url":"https://assets-eu.researchsquare.com/files/rs-6626039/v1/b4c50f475998553bf5fb1cde.png"},{"id":87219342,"identity":"6964fd2b-82fe-408f-b138-672bd665bab4","added_by":"auto","created_at":"2025-07-21 16:03:58","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3202188,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6626039/v1/9b5ba3af-04a3-4096-9ecd-f18e6562fdf8.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Role of laccase and xylanase, with or without ferulic acid esterase-producing Lactiplantibacillus plantarum, on the aerobic stability, protease activity, microbial composition and in vitro degradability of mulberry silage","fulltext":[{"header":"Introduction","content":"\u003cp\u003eChina has long experienced a shortage of high - quality feed resources, especially high-quality protein feeds. This situation increases feed costs and reduces the efficiency of the livestock industry [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Therefore, developing new natural resources, such as nutrient - rich woody plants, is crucial for addressing the feed shortage resulting from increased animal husbandry. Mulberry (\u003cem\u003eMorus alba\u003c/em\u003e L.) is rich in protein, minerals, bioactive substances, and other nutrients and can be considered a new resource for replacing traditional ruminant feeds and alleviating the shortage of traditional protein raw materials [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Moreover, it can improve animal health and the quality of products. For example, it can increase the amino acid and fatty acid content in meat, enhancing the nutritional value and market competitiveness of meat products [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eHowever, mulberry silage lacks lactic acid bacteria (LABs) and has a high buffering capacity and lignocellulose level, leading to subpar ensiling results [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Inoculation with LABs can improve fermentation, inhibit the growth of harmful microbes such as yeasts and moulds and increase the aerobic stability of silage [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. The addition of cellulase and LABs can increase the quality of mulberry silage, conserve more nutrients and increase the antioxidant ability of the feed [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Using FAE-producing LABs in silage can promote lignocellulose decomposition and the release of free ferulic acid, enhancing silage quality and antioxidant properties [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Moreover, our team reported that the use of FAE-producing L. plantarum in combination with lignocellulose hydrolase significantly improved the fermentation quality of papyrus silage and increased the lignocellulosic content in Broussonetia papyrifera [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eLignocellulose consists of lignin, cellulose, and hemicellulose linked by various chemical bonds, forming a complex matrix [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Lignin and hemicellulose increase the difficulty of cellulose degradation by forming lignin‒carbohydrate complexes around the cellulose [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Xylan, a hemicellulose, is located in the secondary wall and is the second most abundant plant polysaccharide [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Xylanase can degrade xylan into monosaccharides and sever lignin‒carbohydrate bonds [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Laccase is a copper-containing phenol oxidase that can oxidize lignin, form high-potential free radicals, and degrade lignin by opening the aromatic ring, resulting in an increase in reducing-sugar recovery during saccharification [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. In silage, laccases can create anaerobic conditions for LAB growth, reducing storage losses from plant cell respiration and aerobic microbe metabolism [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. FAE can break lignin‒hemicellulose linkages, increase the accessibility of degrading enzymes to lignocellulose, and improve the lignocellulose degradation rate and the FAE product can break the linkages between lignin and structural polysaccharides, thereby increasing the in vitro degradability of lignocellulose [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. The combined use of these three enzymes may increase the rate of degradation of the lignin-carbohydrate complex and promote the release of ferulic acid, which has strong antioxidant and antibacterial properties [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. However, the combined use of these three enzymes in mulberry silage and their aerobic stability have not been reported. Therefore, whether the combination of FAE-producing \u003cem\u003eL. plantarum\u003c/em\u003e with laccase and xylanase can have a positive effect on the aerobic stability of mulberry silage needs to be explored.\u003c/p\u003e \u003cp\u003eThis study aimed to examine the synergistic effects of laccase, xylanase, and FAE-producing \u003cem\u003eL. plantarum\u003c/em\u003e on mulberry silage by exploring the aerobic stability, protease activity, microbial composition, in vitro degradability, fermentation characteristics, antioxidant activity and microbial communities during aerobic exposure, thereby providing a basis for the preservation of mulberry.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eRaw materials and additives\u003c/h2\u003e \u003cp\u003eThe mulberry (second crop, tasseling stage) was harvested on 8 September 2023 at the experimental site in Huaxi District, Guiyang City, Guizhou Province. The FAE-producing \u003cem\u003eL. plantarum\u003c/em\u003e used in this study were obtained from a previous isolation by our research team [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. xylanase (X, 100 U mg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) was obtained from Shanghai Macklin Biochemical Technology Co., Ltd and laccase (L, 10 U mg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) was obtained from Beijing Xiasheng Biotechnology Development Co., Ltd.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003ePreparation of silage and aerobic stability analysis\u003c/h3\u003e\n\u003cp\u003ePreparation of mulberry silage, the treatment groups were: Control group (CK) without the addition of bacteria and enzymes, Experimental group (LP) with the addition of FAE-producing strain \u003cem\u003eL. plantarum\u003c/em\u003e at an addition of 1 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e CFU/g FW, the experimental group (LX) with the addition of 25 U/g\u0026thinsp;+\u0026thinsp;25 U/g FW laccase and xylanase, and the experimental group ((M) with the addition of FAE-producing \u003cem\u003eL. plantarum\u003c/em\u003e, laccase and xylanase, respectively, and set up 4 replicates for each treatment. The bags were filled with 400 g/bag and then vacuum sealed for silage (store at room temperature away from light), the bags were opened after 60 days of silage and samples were taken at 0, 1, 3, 5 or 7 days after aerobic exposure to analyze the nutritional quality, fermentation quality and aerobic stability of each treatment, as well as in vitro fermentation after 60 days of fermentation.\u003c/p\u003e \u003cp\u003eAfter opening the bag, the feed is exposed to the air and covered with gauze to prevent contamination by other impurities such as fruit flies and moisture dissipation, and the gas can freely enter the silage bag. Multiple probes of the multi-channel temperature recorder are placed in the center of the feed, and three blanks are set at the same time, and the temperature is recorded at 30-minute intervals. If the sample temperature is higher than the ambient temperature by 2\u0026deg;C, it means that the silage feed is beginning to rot and deteriorate, and the time is recorded [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e].\u003c/p\u003e\n\u003ch3\u003eFermentation parameters and chemical composition\u003c/h3\u003e\n\u003cp\u003eThe fermented mulberry silage samples were opened and mixed well, 10 g of each sample was weighed and put into a self-sealing bag, then 90 mL of sterile water was added, and leaching was carried out at 4℃ for 24 h, finally it was filtered through 4 layers of gauze to obtain the leachate of silage samples. It was stored in the refrigerator at -20℃ and then used to determine the pH value, organic acid and ammoniacal nitrogen content. The pH was determined using a pH meter (PHSJ-5T, INESA Scientific Instrument Co., Ltd., Shanghai, China). Organic acids, such as lactic acid (LA), acetic acid (AA), propionic acid (PA) and butyric acid (BA) were measured by liquid chromatography (Vanquish Core, Thermon Fisher Scientific, US) (KC-811 column; oven temperature: 50\u0026deg;C; flow rate: 1 mL min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) as the method of Li [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Ammoniacal nitrogen (NH₃-N) was determined using the phenol- sodium hypochlorite colorimetric method [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe 200 g of silage samples were taken and dried in an oven at 65℃ until constant weight to determine the dry matter (DM) content [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. The dried samples were pulverized and sealed for storage. Crude protein (CP) was determined by the Kjeldahl method (VAPODEST500, C. Gerhardt GmbH \u0026amp; Co. KG, Germany) [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Neutral detergent fiber (NDF), acid detergent fiber (ADF), and acid lignin (ADL) were determined by the paradigm washed fiber method (ANKOM\u003csup\u003eDELTA\u003c/sup\u003e, ANKOM Technology, Macedon, NY, USA) [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Water-soluble carbohydrates (WSC) were determined by the enthrone- sulfate colorimetric method [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e].\u003c/p\u003e\n\u003ch3\u003eProtein hydrolase activity\u003c/h3\u003e\n\u003cp\u003eUpon the opening of the bag, 10 g of mulberry was sampled from each bag and homogenized with 90 mL of pre - cooled 0.1 M sodium phosphate buffer (pH 6.0, containing 5 mM sodium thiosulfate) for 60 s. The resulting homogenates were filtered through four layers of cheesecloth and subsequently centrifuged at 8,000 \u0026times; g for 10 min at 4\u0026deg;C. The supernatant samples were stored at \u0026minus;\u0026thinsp;80\u0026deg;C for subsequent analysis of protease activities. The activities of aminopeptidase, carboxypeptidase, and acid protein - degrading enzyme were determined in accordance with the method described by Li [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e].\u003c/p\u003e\n\u003ch3\u003eAnalyses of microbial counts\u003c/h3\u003e\n\u003cp\u003eThe bacterial composition was analyzed using the plate counting method by mixing 20 g of sample with 180 mL of distilled water, shaking thoroughly, and performing serial dilutions (10⁻\u0026sup1; to 10⁻⁷). Lactic acid bacteria were enumerated using De Man, Rogosa, and Sharpe (MRS) agar plates (Land Bridge, Beijing, China), which were incubated under anaerobic conditions at 37\u0026deg;C for 48 h. Coliform bacteria were enumerated using Erythromycin Agar plates (Land Bridge, Beijing, China), which were incubated at 30\u0026deg;C for 48 h. Yeasts were enumerated using Bengal Red Agar plates (Land Bridge, Beijing, China), which were incubated at 30\u0026deg;C for 48 h.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eMicrobial community analysis\u003c/h2\u003e \u003cp\u003eAnother 10 g of sample was divided into sealed bags and stored at -80\u0026deg;C in a freezer for the identification of the microorganisms in the sample. To further elucidate the dynamic changes in microorganisms during silage fermentation, DNA was extracted from the samples using the standard cetyltrimethylammonium bromide (CTAB) method for sequencing analysis and strain identification. Polymerase chain reaction (PCR) amplification was conducted with specific primers, using Barcode and GC Buffer from New England Biolabs (Ipswich, MA, USA), as well as high-fidelity and high-efficiency enzymes. The bacterial 16S rRNA gene was amplified using the primers F (5'-CCTAYGGGRBGCASCAG-3') and R (5'-GGACTACNNGGGTATCTAAT-3'). The fungal ITS rRNA gene was amplified using the primers F (5'-CTTGGTCATTTAGAGGAAGTAA-3') and R (5'-GCTGCGTTCTTCATCGATGC-3'). The bacterial 16S amplification protocol was as follows: initial denaturation at 95\u0026deg;C for 2 minutes, followed by 25 cycles of denaturation at 95\u0026deg;C for 30 s, annealing at 55\u0026deg;C for 30 s, and extension at 72\u0026deg;C for 30 s, followed by a final extension was performed at 72\u0026deg;C for 5 minutes. Small fragment libraries were constructed on the basis of the characteristics of the amplified regions; to determine the species composition of the samples, species annotation and abundance analysis of the validated data were performed by Novogene Technology Co., Ltd (Inner Mongolia, China). Subsequently, alpha diversity and coverage value analyses were conducted using the Magic platform (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://magic.novogene.com/\u003c/span\u003e\u003cspan address=\"https://magic.novogene.com/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) to assess differences in community structure.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eIn vitro rumen fermentation trial\u003c/h3\u003e\n\u003cp\u003eRumen fluid was collected and filtered through four layers of gauze into preheated anaerobic thermos flasks and stored until use. In vitro digestion medium was prepared according to the methods of Zhou et al [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Mulberry feed samples were reweighed [1.00 g dry matter] into polyester bags, sealed, and placed in 250 ml fermentation flasks. Two hundred milliliters of the rumen fluid mixture was subsequently dispensed into the preheated flasks at 39\u0026deg;C, and the flasks were closed with plastic caps equipped with one-way valves to prevent the accumulation of fermentation gases after the flasks were filled with CO\u003csub\u003e2\u003c/sub\u003e. The flasks were then transferred to a shaker incubator and shaken at 125 r/min for 72 h at 39\u0026deg;C. After 72 h of incubation, the nylon bags were removed and rinsed with water. The bags were then dried at 65\u0026deg;C until a constant weight was reached. In vitro DM digestibility (IVDMD), NDF digestibility (IVNDFD), and ADF digestibility (IVADFD) were calculated from the change in weight after the reaction.\u003c/p\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eMultifactorial analysis of variance (ANOVA) (one-way or two-way) was performed mainly on the fixed effects of additives and Aerobic exposure time using SPSS 21 software (IBM Corp., New York, NY, USA). Microbial data were normalized by log\u003csub\u003e10\u003c/sub\u003e-transformation on a fresh weight basis. Duncan's and Tukey\u0026rsquo;s multiple comparison was used to determine the statistical difference between the means. Differences were considered significant when \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eChemical composition of raw material before ensiling\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eChemical composition and protein hydrolase activity of ensiling materials.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"2\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eParameter\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMean\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003epH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e6.64\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDM (g/kg FM)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e410.94\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWSC (g/kg DM)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e111.97\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCP (g/kg DM)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e115.43\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNDF (g/kg DM)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e409.24\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eADF (g/kg DM)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e275.98\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eADL (g/kg DM)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e72.75\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAminopeptidase (units h\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e DM of forage)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e17.32\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCarboxypeptidase (\u0026micro;mol of free amino acid released h\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e DM of forage)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e6.95\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAcid proteinase (units h\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e DM of forage)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e69.03\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eDM, dry matter; FM, fresh matter; WSC, water-soluble carbohydrates; CP, crude protein.; NDF, neutral detergent fibers; ADF, acid detergent fiber; ADL, acid detergent lignin.\u003c/p\u003e \u003cp\u003eAs shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, before silage, the pH value of mulberry was 6.64, and the DM content reached 410.94 g/kg fresh matter (FM). The CP content was 115.43 g/kg DM, the NDF content was 409.24 g/kg DM, the ADF content was 275.98 g/kg DM, and the ADL content was 72.75 g/kg DM. Before ensiling, the aminopeptidase activity in mulberry was 17.32 units h⁻\u0026sup1; g⁻\u0026sup1; DM of forage, the acidic protein hydrolase activity was 69.03 units h⁻\u0026sup1; g⁻\u0026sup1; DM of forage, and the carboxypeptidase activity was 6.95 \u0026micro;mol h⁻\u0026sup1; g⁻\u0026sup1; DM. The mulberry had a WSC content of 111.97 g/kg DM, which met the ensiling requirements.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eFermentation of silage during aerobic exposure\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eChanges in fermentation during aerobic mulberry exposure.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"11\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c11\" colnum=\"11\"\u003e\u003c/div\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eParameter\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eTreatment (T)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"5\" nameend=\"c7\" namest=\"c3\"\u003e \u003cp\u003eDays of aerobic exposure (D)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eSEM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c11\" namest=\"c9\"\u003e \u003cp\u003e\u003cem\u003eP\u003c/em\u003e-value\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDay 0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eDay 1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eDay 3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eDay 5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eDay 7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eD\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003eT \u0026times; D\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003epH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCK\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4.71Ac\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4.83Abc\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4.94Abc\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e5.76Aab\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e6.09Aa\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003e0.087\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003e0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4.20Bb\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4.21Cb\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4.26Cb\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e4.27Bb\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e4.97Ba\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLX\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4.66Ac\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4.68Bbc\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4.73Bab\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e4.76Bab\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e4.74Ba\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4.17Bb\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4.19Cb\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4.21Cb\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e4.20Bb\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e5.53ABa\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003eLA (g/kg DM)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCK\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e40.34Db\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e54.36Ba\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e29.36Dc\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e13.24Cd\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e7.12Ce\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003e0.380\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e69.75Bb\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e89.35Aa\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e56.00Bc\u003c/p\u003e \u003c/td\u003e \u003ctd 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align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.6Ab\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.5ABb\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.9Bb\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1.8Ba\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003e0.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.1Bb\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.2Cb\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.3Bb\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.2Cb\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.7Ca\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLX\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.4Ac\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.4Bc\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.7Abc\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.1Ab\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e2.4Aa\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.1Bc\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.2BCbc\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.3Bb\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.2Cbc\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.7Ca\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eLA, lactic acid; AA, acetic acid; PA, propionic acid; BA, butyric acid; NH\u003csub\u003e3\u003c/sub\u003e\u0026ndash;N, ammoniacal nitrogen; TN: total nitrogen. CK, control; LP, FAE-producing \u003cem\u003eL. plantarum\u003c/em\u003e; LX, laccase and xylanase; M, combination of FAE-producing \u003cem\u003eL. plantarum\u003c/em\u003e, laccase and xylanase. T, Treatment; D, Aerobic exposure days, T \u0026times; D, Interacting Treatment and Days of Aerobic Exposure; SEM, standard error of the mean; \u003csup\u003eA\u0026minus;D\u003c/sup\u003eWithin a column and item, means without a common superscript differed (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05); \u003csup\u003ea\u0026minus;e\u003c/sup\u003eWithin a line, means without a common superscript differed (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003eThe data in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e show that the application of additives significantly reduced the pH of the forage mulberry silage (\u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e). During the first 5 days of aerobic exposure, the pH values of the groups treated with FAE-producing \u003cem\u003eL. plantarum\u003c/em\u003e (LP and M) were significantly lower than those of the groups without \u003cem\u003eL. plantarum\u003c/em\u003e (CK and LX) (\u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e). During aerobic exposure, the LA and AA contents in all the additive treatment groups were consistently significantly greater than those in the control group (\u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e). The LA content increased by 6.46\u0026ndash;44.09 g/kg DM, and the AA content increased by 0.46\u0026ndash;1.13 g/kg DM. In particular, compared with the CK treatment, the laccase and xylanase treatments (LX and M) increased the AA content of mulberry silage by 1.49\u0026ndash;2.68-fold. Additive-treated PA and BA levels were unsatisfactory, possibly due to temperature changes in the air during aerobic exposure. The NH\u003csub\u003e3\u003c/sub\u003e-N content increased with the increasing aerobic exposure time, the treatment groups containing FAE-producing \u003cem\u003eL. plantarum\u003c/em\u003e (LP and M) was significantly lower than that of the treatment groups without the addition of FAE-producing \u003cem\u003eL. plantarum\u003c/em\u003e (LX and CK) (\u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e). The NH\u003csub\u003e3\u003c/sub\u003e-N levels were significantly lower in the LX treatment than in the CK treatment for the first 3 days of aerobic exposure (\u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eImpact of additives on the aerobic stability of mulberry\u003c/h2\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAccording to the temperature pattern during aerobic exposure shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, the temperature of the CK group exceeded the ambient temperature by 2\u0026deg;C from 69\u0026ndash;71 h of aerobic exposure, which indicates that the forage began to deteriorate during this period. When the temperature increased to less than 2\u0026deg;C, the silage was relatively stable and less prone to spoilage. During aerobic exposure, the LX treatment group did not exhibit obvious signs of aerobic spoilage. The LP treatment group first exceeded the ambient temperature by 2\u0026deg;C from 132\u0026ndash;146 h, whereas the M treatment group reached this threshold (2\u0026deg;C above the ambient temperature) between 144\u0026ndash;146 h. This sustained temperature increase indicated the onset of mulberry forage decay. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB, the aerobic stabilization time of the additive-treated groups was significantly longer than that of the CK group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), and the aerobic stability, in descending order, was as follows: LX, M, LP, and CK.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eEffect of additives on the chemical composition of mulberry silage\u003c/h2\u003e \u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea, with increasing aerobic exposure time, the DM content of each treatment group gradually decreased. During the first 5 days of aerobic exposure, the DM content of the M treatment group was significantly greater than that of the other treatment groups (\u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e). This could be attributed to the synergistic effect among laccase, xylanase, and FAE-producing \u003cem\u003eL. plantarum\u003c/em\u003e. Figure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec indicates that the crude protein content gradually decreased as the aerobic exposure time increased. There was no significant difference among the treatments during the first 5 days of aerobic exposure, whereas on the 7th day, the M treatment exhibited significantly greater effects than did the control (\u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e). The additive treatments (LP, LX and M) increased the WSC content by 0.78\u0026ndash;7.0 g.kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e DM. The WSC content of the LP and M groups was significantly greater than that of the FAE-producing \u003cem\u003eL. plantarum\u003c/em\u003e groups (LX and M) (\u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e). The contents of NDF and ADF in the LP, LX and M treatments were significantly lower than those in the CK treatment (\u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed, e, f), especially the LP treatment is the most effective. The additive treatments had significantly lower levels of ADL than controls on days 0 and 7 of aerobic exposure (\u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e), whereas there were no significant differences on days 1\u0026ndash;5 of aerobic exposure (\u003cem\u003eP\u0026thinsp;\u0026gt;\u0026thinsp;0.05\u003c/em\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eEffect of additives on the lignocellulose of mulberry silage\u003c/h2\u003e \u003cp\u003eThere was no significant difference in the cellulose content of mulberry silage during the first 5 days of aerobic exposure, but the additive treatment was significantly lower than CK on the 7th day of aerobic exposure (\u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e), especially the M treatment had the lowest content (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). During aerobic exposure, hemicellulose content of additive treatments was significantly lower than that of CK (\u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e). As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec, and lignin content of additive treatment group was significantly lower than that of control group on days 3\u0026ndash;7 of aerobic exposure (\u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e), and there was no significant difference between additive treatments (\u003cem\u003eP\u0026thinsp;\u0026gt;\u0026thinsp;0.05\u003c/em\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eChanges in the activities of three types of proteases during aerobic exposure of mulberry silage\u003c/h2\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAs depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea, b, c, with increasing aerobic exposure time, the activities of the three proteases increased. During aerobic exposure, the aminopeptidase activities of FAE-producing \u003cem\u003eL. plantarum\u003c/em\u003e (LP and M) treatments were significantly lower than those of CK and LX treatments (\u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e), and the LX treatment was not significantly different from the control on days 1 and 3(\u003cem\u003eP\u0026thinsp;\u0026gt;\u0026thinsp;0.05\u003c/em\u003e). Carboxypeptidase activities of all additive treatments were significantly lower than those of the CK treatment during the first 5 days of aerobic exposure (\u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e) and were not significantly different on day 7. Acidic protein hydrolase activity was significantly higher in LP and M treatments than in CK and LX (\u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e), with no significant difference between CK and LX treatments (\u003cem\u003eP\u0026thinsp;\u0026gt;\u0026thinsp;0.05\u003c/em\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eIn vitro digestibility analysis of mulberry fermentation for 60 days\u003c/h2\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e shows that FAE-producing \u003cem\u003eL. plantarum\u003c/em\u003e (LP and M) treatments increased IVDMD (\u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e), with no significant difference between LX and CK (\u003cem\u003eP\u0026thinsp;\u0026gt;\u0026thinsp;0.05\u003c/em\u003e), and no significant difference between LP and M (\u003cem\u003eP\u0026thinsp;\u0026gt;\u0026thinsp;0.05\u003c/em\u003e). IVNDFD was significantly higher in the LX treatment than in M, which was significantly higher than in the CK and LP treatments (\u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e), and the LP treatment had no significant effect on IVNDFD(\u003cem\u003eP\u0026thinsp;\u0026gt;\u0026thinsp;0.05\u003c/em\u003e). Effects of additive treatments on IVADFD (\u003cem\u003eP\u0026thinsp;\u0026gt;\u0026thinsp;0.05\u003c/em\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eChanges in microbial communities during aerobic exposure of mulberry silage\u003c/h2\u003e \u003cp\u003eAs shown in Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, as the duration of aerobic exposure increased, the LAB population gradually increased. Conversely, yeast and \u003cem\u003eColiform\u003c/em\u003e bacteria started to grow from an initial count of zero. Compared with those in the LX and control groups, significantly lower levels were detected in the LP and M treatments (\u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e). In both the LP and M groups, the LAB population tended to increase with increasing pH. At the commencement of aerobic exposure (day 0), no growth of \u003cem\u003eColiform\u003c/em\u003e bacteria or yeast was detected in any of the treatments. In the CK treatment, the growth of \u003cem\u003eColiform bacteria\u003c/em\u003e and yeast commenced on day 1 of aerobic exposure. The additive treatment significantly reduced the number of yeasts (\u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eChanges in microbial counts during aerobic exposure of silage mulberry\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"11\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c11\" colnum=\"11\"\u003e\u003c/div\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eParameter\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eTreatment (T)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"5\" nameend=\"c7\" namest=\"c3\"\u003e \u003cp\u003eDays of aerobic exposure (D)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eSEM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c11\" namest=\"c9\"\u003e \u003cp\u003e\u003cem\u003eP-value\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDay 0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eDay 1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eDay 3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eDay 5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eDay 7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eD\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003eT \u0026times; D\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003eLAB(lg CFU/gFM)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCK\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5.53Bc\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5.64c\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e6.19Ab\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e6.48Ab\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e7.41Aa\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003e0.021\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4.15Cd\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4.98Cc\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4.92Bc\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e6.15Bb\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e6.93Ba\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLX\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6.09A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e6.20A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e6.07A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e5.47C\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e5.63C\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4.00De\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4.39Dd\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e5.04Bc\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e6.28ABb\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e6.98Ba\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003e\u003cem\u003eColiform\u003c/em\u003e bacteria (lg CFU/gFM)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCK\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5.72Ad\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e6.07Ac\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e6.32Ab\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e7.06Aa\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003e0.013\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5.11Bd\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4.74Bc\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e6.20Ab\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e6.84Ba\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLX\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e4.15Bb\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e5.00Ca\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4.73Bc\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e6.16Ab\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e6.99ABa\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003eYeast (lg CFU/g FM)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCK\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e6.03Ac\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e6.07Ac\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e6.46Ab\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e6.91Aa\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003e0.011\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e6.11Bb\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e6.76Ba\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLX\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4.83B\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4.24Bc\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e6.18Bb\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eLAB, lactic acid bacteria; CK, control; LP, FAE-producing \u003cem\u003eL. plantarum\u003c/em\u003e; LX, laccase and xylanase; M, combination of FAE-producing \u003cem\u003eL. plantarum\u003c/em\u003e, laccase and xylanase. T, Treatment; D, Aerobic exposure days, T \u0026times; D, Interacting Treatment and Days of Aerobic Exposure; SEM, standard error of the mean; \u003csup\u003eA\u0026minus;D\u003c/sup\u003eWithin a column and item, means without a common superscript differed (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05); \u003csup\u003ea\u0026minus;e\u003c/sup\u003eWithin a line, means without a common superscript differed (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003eAs presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e, Component 1 and Component 2 explained 69.29% and 10.33% of the total variance, respectively. After 60 days of ensiling, on the third day of aerobic exposure, the LP, LX, and M feed samples were clearly differentiated from CK. By the seventh day of aerobic exposure, LP and M were distinctly separated from LX and CK. This result demonstrated significant disparities in the microbial communities between inoculated and noninoculated silage.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe microbial community structure depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ea indicates that, following ensiling, in aerobically exposed whole-plant mulberry silage, the bacterial community was dominated by the phyla \u003cem\u003eFirmicutes\u003c/em\u003e and \u003cem\u003eProteobacteria\u003c/em\u003e. The LX treatment significantly increased the relative abundance of \u003cem\u003eFirmicutes\u003c/em\u003e. Conversely, the M treatment led to a notable increase in the relative abundance of \u003cem\u003eProteobacteria\u003c/em\u003e on both day 0 and day 7. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eb, the bacterial composition of mulberry silage is predominantly characterized by the genera \u003cem\u003eLactiplantibacillus\u003c/em\u003e, \u003cem\u003eLentilactobacillus\u003c/em\u003e, and \u003cem\u003eStenotrophomonas\u003c/em\u003e. The introduction of additives led to a reduction in the richness of bacterial species in mulberry silage. \u003cem\u003eLentilactobacillus\u003c/em\u003e represents the dominant microflora in LX silage. LP treatment resulted in a significant increase in the relative abundance of \u003cem\u003eLactiplantibacillus\u003c/em\u003e during the first 3 days of aerobic exposure and a decrease on day 7.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAs depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e, \u003cem\u003eAscomycetes\u003c/em\u003e was the dominant phylum throughout the aerobic phase of mulberry silage fermentation. The additive treatments induced a statistically significant reduction in the relative abundance of \u003cem\u003eIssatchenkia\u003c/em\u003e during the aerobic exposure of mulberry. Notably, within the initial three - day period of aerobic exposure in the LX group, \u003cem\u003eIssatchenkia\u003c/em\u003e was scarcely detectable. Treatment with FAE - producing \u003cem\u003eL. plantarum\u003c/em\u003e (LP and M) increased the relative abundance of \u003cem\u003eKazachstania\u003c/em\u003e on days 3 and 7 of aerobic exposure. The additive treatments increased the relative abundance of \u003cem\u003ePhyllactinia\u003c/em\u003e, especially in the LX treatment group on days 3 and 7 of aerobic exposure. The LX treatment also increased the relative abundance of \u003cem\u003eCladosporium\u003c/em\u003e in mulberry silage on days 3 and 7 of aerobic exposure.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAs depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e, the NH₃\u0026ndash;N content of mulberry silage was negatively correlated with the relative abundance of \u003cem\u003eLactiplantibacillus\u003c/em\u003e within the bacterial community (\u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e). The AA content was positively correlated with the relative abundances of \u003cem\u003eLacticaseibacillus\u003c/em\u003e and \u003cem\u003eLentilactobacillus\u003c/em\u003e (\u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.01\u003c/em\u003e) but negatively correlated with the relative abundance of \u003cem\u003eChryseobacterium\u003c/em\u003e (\u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e). The WSC content was negatively correlated with the relative abundances of \u003cem\u003eEnterobacter\u003c/em\u003e and \u003cem\u003eEscherichia-Shigella\u003c/em\u003e (\u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.01\u003c/em\u003e). Conversely, the WSC content was positively correlated with the relative abundance of \u003cem\u003eDelftia\u003c/em\u003e (\u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.01\u003c/em\u003e). The activity of acidic protein hydrolase was positively correlated (\u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.01\u003c/em\u003e) with the relative abundance of \u003cem\u003eStenotrophomonas\u003c/em\u003e, whereas the relative abundance of Chryseobacterium was negatively correlated (\u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.01\u003c/em\u003e) with that of \u003cem\u003eEnterobacter\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eCompared with corn and oat, which are typical conventional forages, mulberry exhibits higher levels of CP, NDF, ADF, and ADL [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. The high fiber content can restrict the efficiency of mulberry utilization; consequently, fiber degradation is crucial for enhancing the utilization of mulberry silage. In the LP treatment, the NDF and ADF contents were significantly lower, likely because the FAE-producing \u003cem\u003eL. plantarum\u003c/em\u003e degraded hemicellulose [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e], leading to a significantly reduced hemicellulose content in the LP treatment. At the later stage of aerobic exposure, the ADL content in the additive treatment was significantly lower than that in the CK group, which can be attributed to the certain delignification ability of the FAE-producing \u003cem\u003eL. plantarum\u003c/em\u003e [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Cellulase and xylanase hydrolyze plant cell walls to generate fermentable sugars, thereby decreasing the NDF and ADF content in oat silage [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. In this study, the combined treatment of laccase and xylanase had a similar effect. Xylanase breaks the chemical bond between lignin and carbohydrates, feruloyl esterase can cleave the ester bond between lignin and hemicellulose, and laccase degrades lignin. Xylanases hydrolyze hemicelluloses, thereby improving the accessibility of cellulases to cellulose [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. When lignin is degraded, the strong bonds among lignin, cellulose, and hemicellulose are disrupted; as a result, the utilization of cellulose and hemicellulose is enhanced [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. We hypothesized that during the ensiling process, the FAE-producing \u003cem\u003eL. plantarum\u003c/em\u003e degraded lignocellulose [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. The additives were more effective in fiber degradation during the later stages of aerobic exposure, thereby enhancing the effectiveness of mulberry silage during the later stages of aerobic exposure. Laccase and cellulase increase the IVDMD, while lactobacilli increase the IVNDFD. A higher IVDMD was associated with a lower NDF content, whereas the IVDMD was positively correlated with the CP content [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. No substantial variation was detected in the IVADFD. Research has indicated that the molecular structure of carbohydrates can affect the rumen degradation rate of NDF in alfalfa and concentrate-based feed [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. The chemical composition of the additive-treated sorghum silage, including its WSC and CP contents, may be optimal for rumen microbial degradation. This could also indirectly explain why the highest LA content and the lowest pH value were observed in the LP and M treatments. The disparities in IVNDFD between the control and additive treatments could be partly attributed to variations in NDF degradation rates [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAn in-depth study revealed that FAE-producing bacteria play a key role in lowering the pH of silage. This finding was consistent with the results of previous research, indicating that these bacteria could effectively promote lactic acid production and acidification in silage, thus contributing to improving the preservation and quality of silage [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Aerobic spoilage of silage could be considered to have occurred when the pH of the silage increased by 0.5 from the initial value [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. According to pH the mulberry silage control started to spoilage on aerobic day 5, LP and M started to spoilage on day 7, whereas LX remained essentially unaltered during aerobic exposure. The LA content significantly decreased after 1 day of aerobic exposure, which was attributed to the breakdown and utilization of LA by microorganisms such as yeasts and aerobes [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Additive treatments increased the AA content of mulberry silage during aerobic exposure. In silage fermentation, increasing the AA content could increase aerobic stability and maintain storage quality, inhibiting the growth of yeasts and undesirable bacteria and helping LAB become the dominant flora, thereby improving silage fermentation quality [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. This is one of the reasons why the additive treatment improves aerobic stability. Aerobic microorganisms (yeast and aerobic bacteria) oxidize organic acids and WSCs to produce carbon dioxide and water, generating heat during growth and reproduction, which leads to an increase in temperature and results in a decrease in aerobic stability [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. This was most likely because a large amount of AA was produced, which inhibited the growth of moulds and yeasts during aerobic exposure [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. This is because the addition of FAE-producing LABs and degrading enzymes may lead to the generation and retention of more WSCs via the decomposition of lignocellulose and rapid and dominant lactic acid fermentation, which can inhibit the growth of undesirable bacteria during the aerobic stage [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. The WSC content of the LX treatments was not significantly different from that of the control. This may be because the pH value of the silages did not decrease below 4.2, resulting in the continued utilization of sugar by other harmful microorganisms [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eCarboxypeptidases, aminopeptidases and acid proteases are the main plant enzymes involved in protein degradation. These enzymes exhibit different behaviors under different pH and temperature conditions and have different sensitivities to inhibitors. The overall extent of protein hydrolysis in silage is largely dependent on pH, which has a strong influence on the activity and stability of proteases [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. This suggests that the addition of FAE-producing \u003cem\u003eL. plantarum\u003c/em\u003e facilitates the inhibition of protein degradation, thereby reducing the ammoniacal nitrogen content. This may be because the additive accelerates \u003cem\u003eLactobacillus\u003c/em\u003e fermentation and inhibits the activity of spoilage bacteria and the cellular respiration in harvested plant tissues [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e].The higher ammoniacal nitrogen content of the LX treatments was probably due to the higher pH resulting in elevated protease activity and protein degradation. The increase in acid protease activity, in conjunction with an elevated abundance of peptides and free amino acids, strongly suggests the pivotal role of acid proteases in protein hydrolysis. Treatments involving FAE-producing \u003cem\u003eL. plantarum\u003c/em\u003e (LP and M) increase acid protease activity by modulating the pH value.\u003c/p\u003e \u003cp\u003eDuring aerobic exposure, the number of \u003cem\u003eLactiplantibacillus\u003c/em\u003e decreased, which may be due to the proliferation of \u003cem\u003eEnterobacter\u003c/em\u003e during silage fermentation, which converted LA produced by \u003cem\u003eLactiplantibacillus\u003c/em\u003e to AA.Since AA is less acidic than LA, the increase in AA content will increase the pH value of the silage and disrupt the growth environment of Lactiplantibacillus [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e].LX was the dominant bacterium during aerobic exposure, and several studies have shown that inoculation with \u003cem\u003eLentilactobacillus buchneri\u003c/em\u003e significantly improved the aerobic stability of corn silage. LX is the dominant bacterium during aerobic exposure, and the results of several studies have shown that inoculation with \u003cem\u003eLentilactobacillus buchneri\u003c/em\u003e significantly improves the aerobic stability of corn silage. This could also explain why the LX treatment had the best aerobic stabilization results. The Ascomycetes phylum was the dominant phylum throughout the aerobic phase of mulberry silage fermentation, which is in agreement with the previous findings of Liu et al [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Issatchenkia has been identified as the main microorganism responsible for aerobic deterioration of maize and barley silage [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Kazachstania unispora was the main yeast species observed in aerobically deteriorated maize silage one of them [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. Once the silage is exposed to air, aerobic microorganisms use LA as a growth substrate, leading to an increase in the pH of the silage. A high-pH environment favours the growth of filamentous fungi, and the proliferation of filamentous fungi, in turn, causes an increase in silage temperature. This phenomenon explains why the aerobic stability of the additive-treated group was greater than that of the CK group. Lactobacillus stenotrophomonas was positively correlated with AA and has the ability to convert some of the LA to AA under acidic conditions as documented by Muck et al [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. Lactobacillus lentilus increased AA content and thus improved aerobic stability. \u003cem\u003eStenotrophomonas\u003c/em\u003e was positively correlated with acidic proteases, Stenotrophomonas spp. have low nutrient requirements but are considered unfavorable for silage due to their ability to degrade proteins [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e].The relative abundance of Lactiplantibacillus, \u003cem\u003eLentilactobacillus\u003c/em\u003e, \u003cem\u003ePhyllactinia\u003c/em\u003e, \u003cem\u003eCladosporium\u003c/em\u003e and \u003cem\u003eKondoa\u003c/em\u003e was positively correlated (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) with AA levels, which may be due to the fact that these taxa have different metabolic functions. In contrast, the relative abundance of \u003cem\u003eWickhamia\u003c/em\u003e was negatively correlated (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) with AA levels. These findings suggest that these microorganisms may be involved in the aerobic stabilization of mulberry silage.\u003c/p\u003e \u003cp\u003eOverall, the application of additives significantly enhanced the fermentation quality of mulberry silage. The M treatment exhibited a pronounced positive impact on in vitro fermentation. Moreover, these treatments effectively improved the aerobic stability of mulberry silage, with the LX treatment affording the highest degree of stability. The utilization of mulberry as feed not only broadens the spectrum of available feed resources but also enhances feeding efficiency.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study revealed that the addition of FAE-producing \u003cem\u003eL. plantarum\u003c/em\u003e, laccase and xylanase increased the LA and AA contents and decreased the pH of mulberry silage, reducing the proliferation of harmful bacterial strains. The aerobic stability of the LX treatment group was the greatest, and that of the M group was similar to that of the control group. Moreover, the additive treatments reduced the species richness of mulberry silage. \u003cem\u003eLentilactobacillus\u003c/em\u003e became the dominant bacterial genus in the LX treatment group, which might have contributed to the increase in the aerobic stability of the mulberry silage. Overall, the addition of laccase, xylanase and FAE \u003cem\u003eL. plantarum\u003c/em\u003e synergistically improved the fermentation and aerobic stability of mulberry silage.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the National Key Research and Development Program of China (2022YFD1300900), Qian Ke He Ping Tai Ren Cai-BQW[2024]003，Qian Ke He Cheng Guo ([2022] Zhong Dian 005), and GZMARS-Forage Industry Technology System of Guizhou Province.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe raw sequence data have been deposited in the sequence read archive at the NCBI (https://www.ncbi.nlm.nih.gov/) under accession number PRJNA1244789\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eEl Naggar S, El-Mesery H S. Azolla pinnata as unconventional feeds for ruminant feeding. \u003cem\u003eBulletin of the National Research Centre\u003c/em\u003e, 2022, \u003cem\u003e46\u003c/em\u003e(1), 66. https://doi.org/10.1186/s42269-022-00752-w\u003c/li\u003e\n\u003cli\u003eSong F, Li Y, Huang J, Lu W, Guo Z, Deng W. Integrated transcriptomic and proteomic analyses reveal the impact of drought and heat stress combination on Morus alba[J]. Environmental and Experimental Botany, 2024, 228(PA):105988-105988. https://doi.org/ 10.1016/j.envexpbot.2024.105988\u003c/li\u003e\n\u003cli\u003eLiu Y, Xiao Y, Xie J, Peng Y, Li F, Chen C, Yin Y, et al. Dietary supplementation with flavonoids from mulberry leaves improves growth performance and meat quality, and alters lipid metabolism of skeletal muscle in a Chinese hybrid pig. \u003cem\u003eAnimal Feed Science and Technology\u003c/em\u003e, 2022, 285, 115211. https://doi.org/10.1016/j.anifeedsci.2022.115211\u003c/li\u003e\n\u003cli\u003eGuo Y, Huang R, Niu Y, Zhang P, Li Y, Zhang W. Chemical characteristics, antioxidant capacity, bacterial community, and metabolite composition of mulberry silage ensiling with lactic acid bacteria. Frontiers in Microbiology, 2024, 15, 1363256. https://doi.org/10.3389/fmicb.2024.1363256\u003c/li\u003e\n\u003cli\u003eYu Q, Su Y, Xi Y, Rong Y, Long Y, Xie Y, Zheng Y, et al. Comparison of the impacts of cellulase and laccase on fermentation quality, bacterial composition and in vitro degradability of anaerobic cofermentation derived from Sudan grass with mulberry under Lactobacillus plantarum and different lignocellulolytic enzyme inoculation. \u003cem\u003eChemical and Biological Technologies in Agriculture\u003c/em\u003e, 2025, 12(1), 41.https://doi.org/10.1186/s40538-025-00760-8 \u003c/li\u003e\n\u003cli\u003eWang T, Teng K, Cao Y, Shi W, Xuan Z, Zhou J, Zhong J, et al. Effects of Lactobacillus hilgardii 60TS-2, with or without homofermentative Lactobacillus plantarum B90, on the aerobic stability, fermentation quality and microbial community dynamics in sugarcane top silage. \u003cem\u003eBioresource Technology\u003c/em\u003e, 2020, 312, 123600. https://doi.org/10.1016/j.biortech.2020.123600\u003c/li\u003e\n\u003cli\u003eHe Li Wen, Zhou Wei, Wang Cheng, Yang FuYu, Chen XiaoYang, Zhang Qing. Effect of cellulase and Lactobacillus casei on ensiling characteristics, chemical composition, antioxidant activity, and digestibility of mulberry leaf silage. 2019. https://doi.org/10.3168/jds.2019-16468\u003c/li\u003e\n\u003cli\u003eLi F H, Ding Z T, Chen X Z, Zhang Y X, Ke W C, Zhang X, Guo X S, et al. The effects of Lactobacillus plantarum with feruloyl esterase-producing ability or high antioxidant activity on the fermentation, chemical composition, and antioxidant status of alfalfa silage. \u003cem\u003eAnimal Feed Science and Technology\u003c/em\u003e, 2021, 273, 114835. https://doi.org/ 10.1016/j.anifeedsci.2021.114835\u003c/li\u003e\n\u003cli\u003eYu Q, Xu J, Li M, Xi Y, Sun H, Xie Y, Zheng Y, et al. Synergistic effects of ferulic acid esterase‐producing lactic acid bacteria, cellulase and xylanase on the fermentation characteristics, fibre and nitrogen components and microbial community structure of Broussonetia papyrifera during ensiling. Journal of the Science of Food and Agriculture, 2024, 104(6), 3543-3558. https://doi.org/10.1002/jsfa.13239\u003c/li\u003e\n\u003cli\u003eQu J, Tian Z, Zhang F, Si C, Ji X. Effect of ultrasound-assisted xylanase pretreatment on the soluble substances of poplar wood and its model construction. \u003cem\u003eAdvanced Composites and Hybrid Materials\u003c/em\u003e, 2024, 7(3), 77. https://doi.org/ 10.1007/s42114-024-00871-0\u003c/li\u003e\n\u003cli\u003eZhang Y C, Wang X K, Lin Y L, Zheng Y L, Ni K K, Yang F Y. Effects of microbial inoculants on fermentation quality and aerobic stability of paper mulberry silages prepared with molasses or Cellulase. Fermentation, 2022, 8(4), 167. \u003cu\u003e https://doi.org/10.3390/fermentation8040167\u003c/u\u003e\u003c/li\u003e\n\u003cli\u003eLiu W, Si Q, Sun L, Wang Z, Liu M, Du S, Jia Y, et al. Effects of cellulase and xylanase addition on fermentation quality, aerobic stability, and bacteria composition of low water-soluble carbohydrates oat silage. Fermentation, 2023, 9(7), 638. https://doi.org/ 10.3390/fermentation9070638\u003c/li\u003e\n\u003cli\u003eXu Q, Zhong H, Zhou J, Wu Y, Ma Z, Yang L, Li X, et al. Lignin degradation by water buffalo. Tropical animal health and production, 2021, 53(3), 344. https://doi.org/10.1007/s11250-021-02787-z \u003c/li\u003e\n\u003cli\u003eZhang S, Dong Z, Shi J, Yang C, Fang Y, Chen G, Tian C, et al. Enzymatic hydrolysis of corn stover lignin by laccase, lignin peroxidase, and manganese peroxidase. \u003cem\u003eBioresource Technology\u003c/em\u003e, 2022, 361, 127699. https://doi.org/ 10.1016/j.biortech.2022.127699\u003c/li\u003e\n\u003cli\u003eBao X, Feng H, Guo G, Huo W, Li Q, Xu Q, Chen, L, et al. Effects of laccase and lactic acid bacteria on the fermentation quality, nutrient composition, enzymatic hydrolysis, and bacterial community of alfalfa silage. \u003cem\u003eFrontiers in Microbiology\u003c/em\u003e, 2022,\u003cem\u003e \u003c/em\u003e13,\u003cem\u003e \u003c/em\u003e1035942. https://doi.org/10.3389/fmicb.2022.1035942\u003c/li\u003e\n\u003cli\u003eJin L, Duniere L, Lynch J P, McAllister T A, Baah J, Wang Y. Impact of ferulic acid esterase producing lactobacilli and fibrolytic enzymes on conservation characteristics, aerobic stability and fiber degradability of barley silage. \u003cem\u003eAnimal Feed Science and Technology\u003c/em\u003e, 2015, 207, 62-74. https://doi.org/ 10.1016/j.anifeedsci.2015.06.011\u003c/li\u003e\n\u003cli\u003eKrueger N A, Adesogan A T, Staples C R, Krueger W K, Dean D B, Littell R C. The potential to increase digestibility of tropical grasses with a fungal, ferulic acid esterase enzyme preparation. Animal Feed Science and Technology, 2008, 145(1-4), 95-108. https://doi.org/ 10.1016/j.anifeedsci.2007.05.042\u003c/li\u003e\n\u003cli\u003ePark C H, Yeo H J, Baskar T B, Park Y E, Park J S, Lee S Y, Park S U. In vitro antioxidant and antimicrobial properties of flower, leaf, and stem extracts of Korean mint. Antioxidants, 2019, 8(3), 75. https://doi.org/10.3390/ antiox8030075\u003c/li\u003e\n\u003cli\u003eYu Q, Li M, Zhang Y, Xu J, Li P, Sun H, Chen C, et al. Effects of different cutting stages and additives on the fermentation quality and microbial Community of Sudangrass (Sorghum sudanense Stapf.) silages. Fermentation, 2023, 9(8), 777. https://doi.org/10.3390/fermentation9080777\u003c/li\u003e\n\u003cli\u003eRanjit N K, Kung Jr L. The effect of Lactobacillus buchneri, Lactobacillus plantarum, or a chemical preservative on the fermentation and aerobic stability of corn silage[J]. Journal of Dairy Science, 2000, 83(3): 526-535. https://doi.org/10.3168/jds.S0022-0302(00)74912-5\u003c/li\u003e\n\u003cli\u003eLi F, Ding Z, Ke W, Xu D, Zhang P, Bai J, Guo X, et al. Ferulic acid esterase-producing lactic acid bacteria and cellulase pretreatments of corn stalk silage at two different temperatures: Ensiling characteristics, carbohydrates composition and enzymatic saccharification. \u003cem\u003eBioresource Technology\u003c/em\u003e, 2019, 282, 211-221. https://doi.org/10.1016/j.biortech.2019.03.022\u003c/li\u003e\n\u003cli\u003eKleinschmit D H, Schmidt R J, Kung Jr L. The effects of various antifungal additives on the fermentation and aerobic stability of corn silage. \u003cem\u003eJournal of dairy science\u003c/em\u003e, 2005, 88(6), 2130-2139. https://doi.org/ 10.3168/jds.S0022-0302(05)72889-7\u003c/li\u003e\n\u003cli\u003eAOAC. Official methods of analysis, 15th ed. AOAC, Arlington, VA.[J]. 1990.\u003c/li\u003e\n\u003cli\u003eVan Soest P V, Robertson J B, Lewis B A. Methods for dietary fiber, neutral detergent fiber, and nonstarch polysaccharides in relation to animal nutrition. \u003cem\u003eJournal of dairy science\u003c/em\u003e, 1991, 74(10), 3583-3597. https://doi.org/10.3168/jds.S0022-0302(91)78551-2\u003c/li\u003e\n\u003cli\u003eOwens V N, Albrecht K A, Muck R E, Duke S H. Protein degradation and fermentation characteristics of red clover and alfalfa silage harvested with varying levels of total nonstructural carbohydrates. \u003cem\u003eCrop Science\u003c/em\u003e, ,1999, 39(6), 1873-1880. https://doi.org/10.2135/cropsci1999.3961873x\u003c/li\u003e\n\u003cli\u003eLi X, Tian J, Zhang Q, et al. Effects of mixing red clover with alfalfa at different ratios on dynamics of proteolysis and protease activities during ensiling[J]. Journal of Dairy Science, 2018, 101(10): 8954-8964. https://doi.org/10.3168/jds.2018-14763\u003c/li\u003e\n\u003cli\u003eZhou Y Q, Cheng Y F, Zhu W Y. The Process and Matters Needing Attention of in vitro Fermentation of Rumen Microorganisms, 2021, Bio-101 e2003663. https://doi.org/10.21769/BioProtoc.2003663\u003c/li\u003e\n\u003cli\u003eCelis-Alvarez M D, L\u0026oacute;pez-Gonz\u0026aacute;lez F, Mart\u0026iacute;nez-Garc\u0026iacute;a C G, Estrada-Flores J G, Arriaga-Jord\u0026aacute;n, C. M. Oat and ryegrass silage for small-scale dairy systems in the highlands of central Mexico. Tropical Animal Health and Production, 2016, 48, 1129-1134. https://doi.org/10.3168/jds.2007-0358\u003c/li\u003e\n\u003cli\u003eFerrero F, Tabacco E, Borreani G. \u003cem\u003eLentilactobacillus hilgardii\u003c/em\u003e inoculum, dry matter contents at harvest and length of conservation affect fermentation characteristics and aerobic stability of corn silage. \u003cem\u003eFrontiers in Microbiology\u003c/em\u003e, 2021, 12, 675563. https://doi.org/10.3389/fmicb.2021.675563\u003c/li\u003e\n\u003cli\u003eZhao J, Dong Z, Li J, Chen L, Bai Y, Jia Y, Shao T. Ensiling as pretreatment of rice straw: The effect of hemicellulase and Lactobacillus plantarum on hemicellulose degradation and cellulose conversion. \u003cem\u003eBioresource technology\u003c/em\u003e, 2018, 266, 158-165. https://doi.org/ 10.1016/j.biortech.2018.06.058\u003c/li\u003e\n\u003cli\u003eLi M, Zhang L, Zhang Q, Zi X, Lv R, Tang J, Zhou H. Impacts of citric acid and malic acid on fermentation quality and bacterial community of cassava foliage silage. \u003cem\u003eFrontiers in Microbiology\u003c/em\u003e, 2020, 11, 595622. https://doi.org/10.3389/fmicb.2020.595622\u003c/li\u003e\n\u003cli\u003e\u0026Ouml;hgren K, Bura R, Saddler J, Zacchi G. Effect of hemicellulose and lignin removal on enzymatic hydrolysis of steam pretreated corn stover. \u003cem\u003eBioresource technology\u003c/em\u003e, 2007, 98(13), 2503-2510. https://doi.org/10.1016/j.biortech.2006.09.003\u003c/li\u003e\n\u003cli\u003eXu T, Du H, Liu H, Liu W, Zhang X, Si C, Liu P, Zhang K. Advanced nanocellulose-based composites for flexible functional energy storage devices. Adv Mater, 2021, 33:2101368. https://doi.org/ 10.1002/adma.202101368\u003c/li\u003e\n\u003cli\u003eLema M, Felix A, Salako S, Bishnoi U. Nutrient content and in vitro dry matter digestibility of silages made from various grain sorghum and sweet sorghum cultivars. Journal of Sustainable Agriculture, 2000, 17(1), 55-70. https://doi.org/10.1080/09712119.2001.9706717\u003c/li\u003e\n\u003cli\u003eZhu J, Li F, Wang Z, Shi H, Wang X, Huang Y, Li S. Effect of Anaerobic Calcium Oxide Alkalization on the Carbohydrate Molecular Structures, Chemical Profiles, and Ruminal Degradability of Rape Straw. Animals, 2023, 13(15), 2421. https://doi.org/10.3390/ani13152421\u003c/li\u003e\n\u003cli\u003eWang S, Li J, Dong Z, Chen L, Shao T. Inclusion of alfalfa improves nutritive value and in vitro digestibility of various straw\u0026ndash;grass mixed silages in Tibet. Grass and Forage Science, 2018, 73(3), 694-704. https://doi.org/10.1111/gfs.12365\u003c/li\u003e\n\u003cli\u003eHeinritz S N, Martens S D, Avila P, Hoedtke S. The effect of inoculant and sucrose addition on the silage quality of tropical forage legumes with varying ensilability. \u003cem\u003eAnimal Feed Science and Technology\u003c/em\u003e, 2012, 174(3-4), 201-210. https://doi.org/ 10.1016/j.anifeedsci.2012.03.017\u003c/li\u003e\n\u003cli\u003eLiu Q, Zhang J, Shi S, Sun Q. The effects of wilting and storage temperatures on the fermentation quality and aerobic stability of stylo silage. Animal Science Journal, 2011, 82(4), 549-553. https://doi.org/10.1111/j.1740-0929.2011.00873.x\u003c/li\u003e\n\u003cli\u003eLiu Y, Li Y, Lu Q, Sun L, Du S, Liu T, Jia Y, et al. Effects of lactic acid bacteria additives on the quality, volatile chemicals and microbial community of leymus chinensis silage during aerobic exposure. Frontiers in Microbiology, 2022, 13, 938153. https://doi.org/ 10.3389/fmicb.2022.938153\u003c/li\u003e\n\u003cli\u003eWu B, Ai J, Li T, Qin W, Hu Z, Siqin T, Niu H, et al. Fermentation quality, aerobic stability, and microbiome structure and function of Caragana korshinskii silage inoculated with/without Lactobacillus rhamnosus or Lactobacillus buchneri. Frontiers in Sustainable Food Systems, 2023, 7, 1255936. https://doi.org/10.3389/fsufs.2023.1255936\u003c/li\u003e\n\u003cli\u003eMugabe W, Shao T, Li J, Dong Z, Yuan X. Effect of hexanoic acid, Lactobacillus plantarum and their combination on the aerobic stability of napier grass silage. \u003cem\u003eJournal of Applied Microbiology\u003c/em\u003e, 2020, 129(4), 823-831. https://doi.org/ 10.1111/jam.14650\u003c/li\u003e\n\u003cli\u003eBing W, Hailing L. Effects of mulberry leaf silage on antioxidant and immunomodulatory activity and rumen bacterial community of lambs[J]. BMC Microbiology,2021,21(1):250-250. https://doi.org/ 10.1186/s12866-021-02311-1\u003c/li\u003e\n\u003cli\u003eWang Y, He L, Xing Y, Zheng Y, Zhou W, Pian R, et al. Dynamics of bacterial community and fermentation quality during ensiling of wilted and unwilted moringa oleifera leaf silage with or without lactic acid bacterial inoculants. \u003cem\u003emSphere\u003c/em\u003e, 2019, 4(4), e00341-19. https://doi.org/ 10.1128/mSphere.00341-19\u003c/li\u003e\n\u003cli\u003eGuo G, Yuan X, Li L, Wen A, Shao T. Effects of fibrolytic enzymes, molasses and lactic acid bacteria on fermentation quality of mixed silage of corn and hulless\u0026ndash;barely straw in the T ibetan Plateau. \u003cem\u003eGrassland Science\u003c/em\u003e, 2014, \u003cem\u003e60\u003c/em\u003e(4), 240-246. https://doi.org/10.1111/grs.12060\u003c/li\u003e\n\u003cli\u003eLiu M, Sun L, Wang Z, Ge G, Jia Y, Du S. Effects of alfalfa hay to oat hay ratios on chemical composition, fermentation characteristics, and fungal communities during aerobic exposure of fermented total mixed ration. \u003cem\u003eFermentation\u003c/em\u003e, 2023, \u003cem\u003e9\u003c/em\u003e(5), 480. https://doi.org/ 10.3390/fermentation9050480\u003c/li\u003e\n\u003cli\u003eZhao J, Dong Z, Li J, Chen L, Bai Y, Jia Y, Shao T. Ensiling as pretreatment of rice straw: The effect of hemicellulase and Lactobacillus plantarum on hemicellulose degradation and cellulose conversion. \u003cem\u003eBioresource technology\u003c/em\u003e, 2018, 266, 158-165. https://doi.org/ 10.1016/j.biortech.2018.06.058\u003c/li\u003e\n\u003cli\u003ePeng S, Xie L, Cheng Y, Wang Q, Feng L, Li Y, Sun Y, et al. Effect of Lactiplantibacillus and sea buckthorn pomace on the fermentation quality and microbial community of paper mulberry silage. \u003cem\u003eFrontiers in Plant Science\u003c/em\u003e, 2024, 15, 1412759. https://doi.org/10.3389/fpls.2024.1412759 \u003c/li\u003e\n\u003cli\u003eLu Q, Wang Z, Sa D, Hou M, Ge G, Wang Z, Jia Y. The potential effects on microbiota and silage fermentation of alfalfa under salt stress. Frontiers in microbiology, 2021, 12, 688695. https://doi.org/10.3389/fmicb.2021.688695\u003c/li\u003e\n\u003cli\u003eMuck R E, Nadeau E M G, McAllister T A, Contreras-Govea F E, Santos M C, Kung Jr L. Silage review: Recent advances and future uses of silage additives. \u003cem\u003eJournal of dairy science\u003c/em\u003e, 2018, 101(5), 3980-4000. https://doi.org/10.3168/jds.2017-13839\u003c/li\u003e\n\u003cli\u003eMu L, Wang Q, Wang Y, Zhang Z. Effects of cellulase and xylanase on fermentative profile, bacterial diversity, and in vitro degradation of mixed silage of agro-residue and alfalfa. Chemical and Biological Technologies in Agriculture, 2023, 10(1), 40. https://doi.org/10.1186/s40538-023-00409-4\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-microbiology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mcro","sideBox":"Learn more about [BMC Microbiology](http://bmcmicrobiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/mcro","title":"BMC Microbiology","twitterHandle":"#bmcmicrobiology","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Mulberry ensiling, Ferulic acid esterase, Lactic acid bacteria, Laccase and xylanase, Aerobic stability, Bacterial community","lastPublishedDoi":"10.21203/rs.3.rs-6626039/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6626039/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eLaccase (L), xylanase (X), and ferulic acid esterase (FAE) act on lignin - carbohydrate complexes. Whether these enzymes, alone or combined, can improve mulberry ensiling and aerobic stability is unclear. This study assessed the effects of L, X, and FAE - producing \u003cem\u003eLactiplantibacillus plantarum\u003c/em\u003e (LP) on whole - plant mulberry silage's fermentation quality, aerobic stability, and microbial communities during aerobic exposure. After 60 days of ensiling, mulberry silage treated with distilled water (CK), LP, laccase\u0026thinsp;+\u0026thinsp;xylanase (LX), or LX\u0026thinsp;+\u0026thinsp;LP (M) was unsealed for 1, 3, 5, or 7 days for exposure to air. The results indicated that the LP and M treatments decreased mulberry silage pH. Lower aminopeptidase and carboxypeptidase activities likely reduced CP degradation and NH₃-N content (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05), while increasing LA and WSC production. Compared with the CK treatment, the addition of LX and M increased the AA content by 1.49-2.68-fold, indicating greater aerobic stability (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), which contributed to maintaining the storage quality of the silages during aerobic exposure. The application of additives to mulberry silage reduced the species richness; specifically, the additive treatments led to an increase in the relative abundance of \u003cem\u003eKondoa\u003c/em\u003e and \u003cem\u003eLentilactobacillus\u003c/em\u003e while decreasing that of \u003cem\u003eEnterococcus\u003c/em\u003e and \u003cem\u003eDelftia\u003c/em\u003e. Notably, \u003cem\u003eLentilactobacillus\u003c/em\u003e exhibited the capacity to inhibit the growth of other harmful microorganisms and emerged as the dominant genus within the LX group. In conclusion, treatment with the combination of laccase, xylanase, and FAE-producing \u003cem\u003eL. plantarum\u003c/em\u003e can serve as an effective method to improve the silage quality and aerobic stability of mulberry.\u003c/p\u003e","manuscriptTitle":"Role of laccase and xylanase, with or without ferulic acid esterase-producing Lactiplantibacillus plantarum, on the aerobic stability, protease activity, microbial composition and in vitro degradability of mulberry silage","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-19 08:21:36","doi":"10.21203/rs.3.rs-6626039/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-06-10T05:18:22+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-06-09T03:22:04+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-06-08T09:26:55+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"224894720262706213375331007839825886568","date":"2025-06-05T07:50:58+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"145265039728995959991264963140941450651","date":"2025-06-05T07:38:39+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"164436757161737504359491514689340466495","date":"2025-06-03T16:00:35+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"223717585059999721285482666835172609326","date":"2025-06-03T09:02:09+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-06-01T02:36:29+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-05-26T09:26:22+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"37158571723140889764019455349005321772","date":"2025-05-25T03:20:34+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"923491712924970210601583160679860155","date":"2025-05-22T11:01:13+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"83686461435772604230258666898665941149","date":"2025-05-21T02:27:56+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"165327225253257968058635563629902008492","date":"2025-05-21T01:53:52+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"72777828944560447807469220561246994526","date":"2025-05-15T05:28:32+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-05-15T01:25:15+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-05-14T11:13:15+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-05-13T14:14:38+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-05-13T14:09:25+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Microbiology","date":"2025-05-09T07:14:37+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-microbiology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mcro","sideBox":"Learn more about [BMC Microbiology](http://bmcmicrobiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/mcro","title":"BMC Microbiology","twitterHandle":"#bmcmicrobiology","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"a5536db8-3d49-4853-ad6d-ada55d9d99dc","owner":[],"postedDate":"May 19th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-07-21T16:00:23+00:00","versionOfRecord":{"articleIdentity":"rs-6626039","link":"https://doi.org/10.1186/s12866-025-04165-3","journal":{"identity":"bmc-microbiology","isVorOnly":false,"title":"BMC Microbiology"},"publishedOn":"2025-07-16 15:57:19","publishedOnDateReadable":"July 16th, 2025"},"versionCreatedAt":"2025-05-19 08:21:36","video":"","vorDoi":"10.1186/s12866-025-04165-3","vorDoiUrl":"https://doi.org/10.1186/s12866-025-04165-3","workflowStages":[]},"version":"v1","identity":"rs-6626039","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6626039","identity":"rs-6626039","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}