Lactobacillus plantarum S1 (LAB) was isolated from the corn stalks, and CDMs with higher CMCase activity sizes were isolated from rotted corn stalks (Song et al., 2017).

The corn stalks (moisture content of 75%) were obtained from Hohhot, Inner Mongolia, China. After natural drying, the corn stalks were divided into two groups. Group 1: the corn stalks were pulverized and passed through a 40-mesh sieve to embed LP S-1 and culture CDM as the substrate. Group 2: the corn stalks were cut into 1–3 cm segments for the ensiling process.

Solid MRS medium: peptone 10 g (AR, Beyotime), beef extract 10 g (AR, Beyotime), yeast extract 5 g (AR, Beyotime), glucose 20 g (AR, TCI), diammonium hydrogen citrate 2 g (AR, Aladdin), Tween 80 1 mL (AR, Aladdin), CH₃COONa 5 g (AR, Beyotime), K₂HPO₄ 2 g (AR, Beyotime), MgSO₄ 0.58 g (AR, Solarbio), MnSO₄ 0.25 g (AR, Solarbio), and agar 18 g (AR, Solarbio) were dissolved in 1 L distilled water and sterilized at 115°C for 35 min after pH was adjusted to 6.2–6.6. Solid cellulose medium: CMC-Na 5 g (AR, Beyotime), agar 20 g, K₂HPO₄ 1 g, MgSO₄ 0.5 g, (NH₄)₂SO₄ 2 g (AR, Beyotime), and NaCl 0.5 g (AR, Beyotime) were dissolved in 1 L distilled water and sterilized at 115°C for 35 min. Liquid corn-stalk medium: 10 g of corn stalks was added into 1 L distilled water and sterilized at 115°C for 35 min. Liquid cellulose medium: CMC-Na 10 g, K₂HPO₄ 1 g, MgSO₄ 0.5 g, (NH₄)₂SO₄ 2 g, NaCl 2.5 g, beef extract 2.5 g, and peptone 5 g were dissolved in 1 L of distilled water and sterilized at 115°C for 35 min. Liquid L. medium: Peptone 5 g, beef extract 5 g, yeast extract 5 g, and glucose 20 g were dissolved in 1 L distilled water and sterilized at 115°C for 35 min.

The isolates of CDM were inoculated into a liquid corn-stalk medium. After 15 days of fermentation at 30°C, the weightlessness and the concentration of soluble sugar were determined to evaluate the ability of degradation of corn-stalk cellulose.

The preparation of lyophilized CDM was divided into two steps: the collection conditions of CDM cells and the conditions of lyophilization of CDM. The single-factor experiment (SFE) and response surface methodology (RSM) were used to optimize the collection of cells and the selection of the best cryoprotectant in the preparation process. First, the effect of centrifugation, the time of centrifugation (0, 10, 20, 30, and 40 min), the temperature of centrifugation (−4, 0, 4, 8, and 10°C), and the rate of rotation (2,000, 4,000, 6,000, and 8,000 r/min) on the survival rate of BM 2–4 were evaluated using the SFE. Moreover, the best condition of cell collection was optimized using the RSM. Second, the effect of cryoprotectant, maltodextrin (5, 10, and 20%), skim milk (5, 10, and 20%), peptone (5, 10, and 20%), trehalose (5, 10, and 20%), and glucose (5, 10 and 20%) on the survival rate of BM 2–4 was evaluated using the SFE, and the composition of cryoprotectant was optimized using RMS. In addition, the process of lyophilization is as follows: the cells of CDM (adding cryoprotectant) were pre-frozen at −20°C after centrifugation and then transferred into a freeze dryer for lyophilizing (−50°C, vacuum pressure of <10 Pa). The number of microbial cells was counted using a hemacytometer, and the microbial survival rate was calculated to evaluate the conditions of lyophilizing CDM.

The preparation process for embedding LP S-1 was described in our previous study. The embedded process consisted of two steps, and the brief process was as follows: LP S-1 was adsorbed with corn-stalk powder, followed by the addition of sodium alginate (1.2%) and CMC-Na (0.5%) to 4% CaCl₂ for 24 h (Song et al., 2018).

Lyophilized BM2-4 and embedded LP S-1 were inoculated into a liquid corn-stalk medium (static cultivation at 30°C) to evaluate the effect of lyophilized BM2-4 on the stability of embedded LP S-1 by recording the residual number of embedded balls of LP S-1 during the fermentation process.

SFE was used to investigate the effect of inoculum size (2, 3, 4, 5, 6, 7, and 8%), fermentation time (0.25, 0.5, 1, 2, 3, 5, 7, and 9 days), moisture content (45, 55, 65, 75, 85, and 95%), and proportion (embedded LP S-1:lyophilized BM2-4, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, and 1:4, g/g) on the concentration of crude fiber (CF), crude protein (CP), and pH in the ensiling process of corn stalks. The mixed fermentation broth (LP S-1 and BM2-4) and embedded LP S-1 were used as a control to evaluate the performance of this slow-release strategy. The experiment was conducted in the anaerobic fermentation bags (50 × 30 cm) at 30°C; CP and CF were determined at the beginning and end of the ensiling process.

The concentration of soluble sugar was determined using the DNS method (Sun et al., 2006), and the survival rate of lyophilization was determined using the method of Shao et al. (2014). The determination of CP and CF was carried out by the description in Chinese National Standards of GB/T 6432–94 and GB/T 6434–2006. pH was determined using a pH meter.

The rate of weightlessness is calculated by Equation 1.

w: The weight of corn stalk after ensiling.

W: The weight of corn stalk before ensiling.

The number of living microbial cells was counted by the method of hemacytometry, and the microbial survival rate was calculated using Equation 2.

m: The number of living microbial cells after treatment per volume.

M: The number of living microbial cells before treatment per volume.

The rate of cellulose degradation is calculated using Equation 3.

c: The concentration of crude fiber after ensiling.

C: The concentration of crude fiber before ensiling.

The rate of protein increase is calculated using Equation 4.

p: The concentration of crude protein before ensiling.

P: The concentration of crude protein after ensiling.

Three replications were performed in all treatments, and a one-way analysis of variance (ANOVA) was used to evaluate the differences between the two groups. The difference between the two groups was regarded as statistically significant if p < 0.05. Design Expert 8.05b was used to design and analyze the RSM. Figures were drawn by Microsoft Office Excel 2023.

In the previous study, 54 CDMs were isolated from rotted corn stalks, and 16 of them with higher CMCase activity were identified (the majority belonging to the genus Bacillus; Song et al., 2017). In this study, the corn-stalk powder was used as the substrate to select the CDM with a higher ability of corn-stalk degradation from the 16 isolates. The results showed that six isolates (6–9, 3–4, 3–1, 14–1, 2–4, and 14–3) had higher weightlessness (Figure 1A), and the weightlessness of them was significantly higher than others (p < 0.05). This indicated that these isolates had a higher ability of corn-stalk degradation. Moreover, a contrast experiment of the six isolates was conducted, and the soluble sugar was determined to further evaluate the ability of cellulose degradation. The results showed that five isolates (3–1, 6–9, 3–4, 14–1, and 2–4) consumed the pre-existing soluble sugar in 0–12 h and began to produce sugar after 12 h of fermentation. The concentration of soluble sugar in the fermentation broth of 3–4 and 2–4 reached a plateau at 48 h, and the concentration of soluble sugar in the fermentation broth of 2–4 (9.54 mg/mL) was significantly higher than other isolates after 96 h of fermentation (p < 0.05, Figure 1B). Therefore, 2–4 was selected as CDM for further study in this study. Many microorganisms can produce cellulase (Lynd et al., 2002). It was reported that the genus Bacillus not only can degrade cellulose (Duman et al., 2016; Saffari et al., 2017; Yadav et al., 2024) but can also inhibit yeasts, molds, and certain harmful microorganisms (Lara et al., 2016; Li et al., 2024), thereby improving the quality of feed and performance of livestock growth (Zhang et al., 2016). B. methylotrophicus has the effect of inhibiting fungal growth (Li et al., 2016; Tumbarski et al., 2018; Jiang et al., 2019) and it may have positive effects on the corn-stalk silage. In this study, 2–4 was identified as Bacillus methylotrophicus (BM 2–4, a facultative anaerobe) in our previous study (Song et al., 2017).

The whole lyophilization process of BM 2–4 was separated into two stages to obtain the maximum survival rate, cell collection, and lyophilization. In the first stage, parameters of cell collection were evaluated (SFE) and optimized (RSM) for collecting the cells of BM 2–4. After SFE, the optimal rates of rotation, centrifugation time, and temperature of centrifugation were 5,000 rpm/min, 10 min, and 4°C, respectively (Figure 2A), and the results of RMS showed that the maximum survival rate was 89.21% at the conditions of 5,600 r/min in 4.4°C for 9.5-min centrifugation (Figure 2B).

In the second stage, the effect of five cryoprotectants (maltodextrin, skim milk, peptone, trehalose, and glucose) on the survival rate of BM 2–4 in the process of lyophilization was investigated. The results showed that skin milk, peptone, and glucose had a positive impact on the survival rate of BM 2–4, with the optimal concentrations being 10.0, 10.0, and 5.0%, respectively (Figure 3A). Furthermore, the predicted highest survival rate of 88.85% was achieved under the conditions of 10.36% skim milk, 10.38% peptone, and 5.64% glucose as verified by RSM (Figure 3B). The concentration of skim milk, peptone, and glucose was adjusted to 10.4, 10.4, and 5.2%, respectively, and the survival rate of 89.53% was achieved using a verification test. Lyophilization is an effective method for preserving the vitality of microorganisms; however, the formation of ice crystals can damage the structure of the cell membrane, leading to decreased vitality and metabolic activity (Beney and Gervais, 2001; Araújo et al., 2020). Cryoprotectants have a positive effect on the microbial survival rate in the lyophilization process and they can protect cells from dehydration (Nunes et al., 2018; Cheng et al., 2022). Skim milk can protect the cells from intense environmental change, and it has been used more widely than other cryoprotectants. Fonte et al. (2015) and Peiren et al. (2015) proved that different kinds of cryoprotectants work together to obtain a higher survival rate after lyophilization. In this study, skim milk, peptone, and glucose were selected as lyoprotectants to improve the survival rate of BM 2–4 in the lyophilization process.

The stability of embedded LP S-1 in the fermentation broth of BM 2–4 was investigated, and the results are shown in Figure 4. After 36 h of fermentation, the number of embedded LP S-1 was decreased from 100 to 0 in static cultivation at 30°C. This indicated that BM 2–4 could release LP S-1 slowly by disrupting the structure of the embedding material. The degradation time of embedded LP S-1 was a key factor in the degradation of corn stalks by BM 2–4. In this study, LP S-1 was embedded in a mixture of materials (corn stalk powder and calcium alginate gel; Song et al., 2018). This setup laid the foundation for destroying the embedded material of LP S-1 by BM 2–4, leading to the production of soluble sugars that stimulate the growth of LP S-1 during the ensiling process. The calcium alginate gel is the most common material to immobilize the enzyme or microorganism. It is reported that the stability of calcium alginate gel immobilization was degraded at the low pH (Simó et al., 2017), and the feature can be advantageous when used as slow-release material in our study. In the medical field, the encapsulation of certain active substances in alginate substrates can protect drugs, prevent premature drug inactivation, delay the release of drugs, and enable drugs to reach the target point at a fixed time to complete targeted therapy. Moreover, calcium alginate gel has been proven to be a safe additive for food (Prevost and Divies, 1992) and is feasible for use in animal feed production.

The effects of inoculum size, proportion of embedded LP S-1 and lyophilized BM2-4, fermentation time, and moisture on the CF and CP of corn-stalk silage were investigated, and the results are shown in Figure 5. Higher degradation of CF and increased CP (p < 0.05) in the ensiling process of corn stalks were obtained under the following conditions: 6% inoculum size (Figure 5A), 7 days of fermentation (Figure 5B), 85% moisture content (Figure 5C), and a 2:1 ratio of embedded LP S-1 and lyophilized BM2-4 (Figure 5D), respectively.

Based on the results of Section 3.4, the mixed fermentation broth (LP S-1 and BM 2–4) and embedded LP S-1 were used as the control (the concentration of cells is the same) to evaluate the effect of inoculating embedded LP S-1 and lyophilized BM 2–4 on CF, CP, and pH at the end of the ensiling process of corn stalk. After 7 days of fermentation, inoculating embedded LP S-1 and lyophilized BM 2–4 can significantly degrade CF by 3.8% and increase CP by 3.7% compared to inoculating the mixed fermentation broth of LP S-1 and BM 2–4 (p < 0.05, Figure 6). In addition, inoculating embedded LP S-1 and lyophilized BM 2–4 had no significant effect on the final pH compared to the mixed fermentation broth of LP S-1 and BM 2–4 (p > 0.05). This indicated that inoculating embedded LP S-1 and lyophilized BM 2–4 has a positive effect on the silage.

In the ensilaging process, LAB can secrete lactic acid and improve the flavor of silage (Liu et al., 2016). However, lactic acid can inhibit the growth and activity of other microorganisms including CDM (Wang Y, et al., 2021). The decline of pH caused by LAB was the main factor inhibiting the growth of CDM and the activity of cellulase. Therefore, the separation of LP S-1 and BM 2–4 to work at different times (BM2-4 first) can improve the degradation of cellulose in the ensiling process of corn stalk. Compared to the two-step process and pretreatment of corn stalks (Yang et al., 2001; Krafft et al., 2020) for silage, the slow-release strategy also can degrade cellulose and pH in the ensilaging process through a one-step operation.