Whole-plant corn (Zea mays L.) silage (WPCS) is the world’s most widely used forage source in dairy cow diets (USDA, 2014). Given its importance, strategies to enhance nutritive value and fermentation profile, reduce DM losses, and increase aerobic stability have been evaluated (Diepersloot et al., 2022). The most commonly adopted strategy to achieve these objectives is to use microbial inoculants, mainly containing lactic acid bacteria (LAB) (Ávila and Carvalho, 2020; Carvalho et al., 2020), which might improve fermentation by accelerating the drop in pH. These bacteria can produce acids, bacteriocins, and other compounds that inhibit undesirable microorganisms, increasing silage aerobic stability (Carvalho et al., 2020).

Obligate heterofermentative LAB can produce antifungal compounds, which can inhibit yeast and mold growth; thus, increasing aerobic stability (Arriola et al., 2021; Drouin et al., 2023). Among the obligatory heterofermentative LAB, the species Lentilactobacillus buchneri (LB) is the most studied silage inoculant (Kung et al., 2018). Arriola et al. (2021) revealed that LB inoculation increased aerobic stability, raised the concentration of 1,2-propanediol and acetic and propionic acids, reduced the lactic acid concentration, and lowered the populations of yeasts and filamentous fungi in several silages (corn, sorghum, temperate and tropical grass, sugarcane, alfalfa, grain, and high moisture corn). The meta-analysis also showed that the effects of LB inoculation were dependent on storage time, with a more pronounced increase in aerobic stability and reduction in yeast population observed after 90 days of storage (Arriola et al., 2021). However, da Silva et al. (2020) observed a reduction in the yeast population and an increase in aerobic stability with the inoculation of LB (40788) after 10 days of storage of high-moisture corn.

An important point to note is that the results depend on the strain used (Carvalho et al., 2020). To our knowledge, no studies have assessed the results of this strain inoculation in silages, warranting further investigation for specific strain intended for commercial use (Carvalho et al., 2020).

Another point for consideration is the need to evaluate the action of obligate heterofermentative strains for different storage periods, because some strains of LB have shown positive effects, even with short storage times (Arriola et al., 2021; Drouin et al., 2023).

The objective of the present study was to evaluate the effect of Lentilactobacillus buchneri, LBu 01 - CGMCC 19250 strain, on nutritive value and fermentation profile of WPCS under different storage periods. We hypothesized that microbial inoculation would be more pronounced acetic acid production and aerobic stability and that these effects would increase with storage length.

Table 1 shows the chemical and microbiological compositions of the control and inoculated forage. Table 2 presents the probability values (P values) for the effects of inoculants, storage time, and their interactions.

Table 1

Item	Control	LB1
pH	5.68	5.61
Dry mattercorr (DM; %)	34.1	34.3
Crude protein (CP; % DM)	6.90	7.40
Soluble protein (% CP)	45.0	45.0
Ammonia (% DM)	0.29	0.31
Starch (% DM)	29.2	28.4
Water-soluble carbohydrates (% DM)	9.8	8.7
Starch digestibility 7 h (% starch)	49.0	50.0
Neutral detergent fiber (NDF) (% DM)	47.9	50.1
In vitro NDF digestibility 30 h (% NDF)	55.0	55.0
Non-digestible NDF 30 h (% DM)	17.9	18.6
In vitro DM digestibility 24 h (% DM)	77.0	76.0
Bacteria in MRS (log CFU/g)	7.06	7.76
Yeasts (log CFU/g)	6.24	5.94
Filamentous fungi (log CFU/g)	5.47	5.51
Bacteria in EMB (log CFU/g)	6.88	6.94
Aerobic spore-forming bacteria (log CFU/g)	5.99	5.71

¹ Strain: LBu01.

Table 2

Item	Inoculant (I)	Storage (S)	I×S
pH	0.03	<0.01	0.87
Dry matter (DM)	<0.01	<0.01	<0.01
DM loss	<0.01	<0.01	<0.01
Crude protein	0.79	0.21	0.90
Soluble protein	0.96	<0.01	0.68
Ammonia	0.63	<0.01	0.13
Starch	0.03	0.55	0.05
Water-soluble carbohydrates	0.62	<0.01	0.26
Starch digestibility 7 h	0.75	0.41	0.81
Neutral detergent fiber (NDF)	0.25	0.14	0.43
In vitro NDF digestibility 30 h	<0.01	<0.01	0.67
Non-digestible NDF 30 h	<0.01	<0.01	0.02
In vitro DM digestibility 24 h	<0.01	<0.01	0.16
Lactic acid	0.11	<0.01	0.03
Acetic acid	0.33	<0.01	0.31
Ethanol	0.55	0.91	0.20
Bacteria in MRS	<0.01	0.04	<0.01
Yeasts	0.15	0.33	0.23
Aerobic spore-forming bacteria	0.23	0.12	0.34

The pH was lower for the inoculated silage (3.68) than for the control silage (3.73). At 15 days of storage, the pH reached its lowest value (3.50), whereas it increased after 60 days (4.00) (Table 3). An interaction between inoculation and storage period was evident for silage DM concentration and DM losses (Table 2). After three days of storage, no difference was detected between the DM concentration of the control silage and the inoculated silage; however, after 15 and 62 days, the DM concentration was greater for inoculated than for control silage (Figure 1A). The DM concentration of the inoculated silage did not vary from three (33.4%) to 15 days of storage (33.9%); however, the DM of the inoculated silage was lower (32.9%) at 62 days than after 15 days of storage. The DM of the control silage decreased consistently from three (33.9%) to 62 (30.1%) days of storage. The percentage of DM losses showed an inverse pattern to the DM concentration (Figure 1B). Dry matter losses were lower in LB silage than in control silage after storage for 15 (6.3% control; 1.6% LB) and 62 (12.9% control; 4.8% LB) days. The DM loss in the inoculated silage increased from 15 to 62 days of storage, whereas the loss in the control silage consistently increased at each period (Figure 1B).

Table 3

Variable	Inoculant		SEM	Storage (days)			SEM
	Control	LB		3	15	62
pH	3.73B	3.68A	0.012	3.61b	3.50c	4.00a	0.014
Crude protein (% DM)	7.33	7.30	0.087	7.20	7.47	7.28	0.107
Soluble protein (% CP)	55.47	55.53	1.000	51.20b	55.20b	60.10a	1.225
Ammonia (% DM)	0.53	0.54	0.012	0.42c	0.55b	0.63a	0.015
Water-soluble carbohydrates (% DM)	4.69	4.45	0.346	6.40a	3.71b	3.60b	0.423
Neutral detergent fiber (NDF) (% DM)	49.23	48.35	0.531	49.29	49.39	47.7	0.650
Starch digestibility 7 h (% starch)	59.8	60.3	1.189	61.5	60.0	58.7	1.457
In vitro NDF digestibility 30 h (% NDF)	54.9B	56.9A	0.463	54.2b	57.3a	56.3a	0.567
In vitro DM digestibility 24 h (% DM)	75.9B	77.2A	0.186	75.5b	76.8a	77.4a	0.227
Acetic acid (% DM)	0.96	1.06	0.063	0.77b	0.85b	1.41a	0.077
Ethanol (% DM)	0.66	0.61	0.052	0.65	0.64	0.62	0.063
Yeasts (log CFU/g)	5.93	5.60	0.152	5.82	5.54	5.94	0.186
Aerobic spore-forming bacteria (log CFU/g)	5.14	5.36	0.126	4.99	5.49	5.27	0.155

The inoculant mean values with different capital letters are significant at P<0.05 according to Tukey’s test. The inoculant mean values with different lowercase letters are significant at P<0.05 according to Tukey’s test.

Figure 1

SEM - DM = 0.295; SEM - DM loss = 0.850.

SEM - standard error of the means.

Capital letters compare the inoculant within storage period; lowercase letters compare the storage periods.

The chemical and microbiological characteristics of the forage examined in the present study are similar to those commonly reported in the literature for corn hybrids used in Brazil (Costa et al., 2021; Rossi et al., 2023). The pH observed in the silage indicate desirable fermentation. The pH of the inoculated silage was slightly lower than that of the control silage highlighting a favorable point for the strain under study. Generally, inoculation of silage with heterofermentative LAB results in an increase in the silage pH (Agarussi et al., 2022; Rossi et al., 2023). However, as observed in this study, depending on the strain used, silages inoculated with Lentilactobacillus buchneri may have a pH equal to or lower than that of uninoculated control silages (Agarussi et al., 2022; Rossi et al., 2023). This result may be related to the metabolism of LBu01 or a change in the diversity of other bacteria naturally present in the silage due to inoculation (Bai et al., 2022; Guo et al., 2023). More detailed studies of the metabolism and effect of LBu01 on microbial diversity should be carried out in the future.

Contrary to the findings of this study, inoculation with heterofermentative strains is associated with the highest percentage of fermentative losses. However, several studies have shown that losses are lower following inoculation with some of these strains than in uninoculated silages or silages inoculated with some homofermentative strains (Arriola et al., 2021; Costa et al., 2021; Agarussi et al., 2022). According to the literature, high pH and DM loss in silages inoculated with heterofermentative LAB strains are explained by a metabolism that produces more acetic acid because this acid has a lower capacity to reduce pH and its production pathway generates CO₂ (Ávila and Carvalho, 2020). Inoculation with strain LBu01 resulted in silages with lower pH and lower DM loss without changing the acetic acid concentration. Arriola et al. (2021) and Agarussi et al. (2022) reported that the losses related to inoculation with LB depend on the dose applied, the type of forage, the combination with other LAB, and the strain of LB used. These observations reinforce the need for the systematic isolation and selection of strains intended for inoculation in silage (Carvalho et al., 2020). An increase in fermentative losses is common throughout the storage period; however, the LBu 01 strain evaluated in this study was able to minimize the losses that occurred in the control silage. This loss reduction is generally associated with a reduced presence of undesirable microorganisms, such as yeast. However, the populations of the undesirable microorganisms studied (yeasts, filamentous fungi, enterobacteria, and ASFB) did not change significantly, but the LAB population increased after 62 days of storage. Some inoculants (Lacticaseibacillus rhamnosus IMI 507023; Lactiplantibacillus plantarum IMI 507026; Pediococcus pentosaceus IMI 507025) can reduce N-NH₃ and soluble protein levels, but this is mainly observed in silages that are predisposed to have high values of these variables (e.g., silages with low DM) (Kung et al., 2018; Gonda et al., 2023). In the present study, the soluble protein concentration increased from 15 to 62 days, and the NH₃ concentration increased at all periods evaluated. For silages, slight increases in ammonia concentration (<15% total N) and soluble protein (<60% total N) during the storage period are acceptable and may reflect the action of plant enzymes and microorganisms. In corn grain silage, an increased soluble protein concentration is associated with prolamin degradation and increased starch digestibility. Although an increase in the soluble protein concentration was observed in the present study, starch digestibility did not increase as the storage period progressed, in agreement with previous findings by Rossi et al. (2023).

The increase in starch concentration observed with the interaction between LB inoculation and longer storage time was unexpected. Drouin et al. (2023) observed an increase in starch concentration after 180 days of storage of high-moisture corn silage, but did not observe an effect of inoculation with L. buchneri NCIMB 40788 on this nutrient. Kok et al. (2024) reported that inoculating Lactococcus lactis and Lentilactobacillus buchneri (SiloSolve FC^®) showed a tendency to increase starch concentration (38.4% DM) compared with silage without inoculation (36.9% DM). However, these previous studies did not discuss the possible causes of their results. Tavares et al. (2024) found that inoculation with a microbial inoculant (Lentilactobacillus buchneri and Lactiplantibacillus plantarum) increased the starch concentration of WPCS (+2.9% DM), and they attributed this increase to the efficiency of the inoculant in preserving nutrients through fermentation. In the present study, we associated the increased in starch content (2.5%) with the lower loss of DM in the inoculated silage. In other studies, in which LB was inoculated alone, the increase in starch concentration was not observed. This may be because no reduction in DM losses was observed with inoculation (Drouin et al., 2023; Rossi et al., 2023).

The in vitro NDF digestibility at 30 h and DM at 24 h were highest in the inoculated silage. Other studies have reported increases in NDF digestibility when L. buchneri was applied alone or combined with whole-plant corn silage (Weinberg et al., 2007; Rossi et al., 2023; Kok et al., 2024). This result can be associated with the production of the ferulic acid esterase (FAE) enzyme. Some strains of L. buchneri are known to produce this enzyme, which hydrolyzes the ferulic acid ester groups present in the bond between hemicellulose and lignin (Muck et al., 2018). The inoculated silages in the present study showed an increase of 3.1 (% NDF) and 1.3 (% DM) in the digestibility of NDF and DM from three to 15 days of storage. Considering the role of LAB in increasing the digestibility of the fibrous fraction, we speculate that FAE requires some time for production and activation. However, the ability of the LBu01 strain used in this study to produce FAE was not evaluated; therefore, future studies should be conducted to confirm this possibility. The increase in DM digestibility observed following LB inoculation may be associated with increased NDF digestibility.

The lactic acid concentrations (means of 7.05 and 6.38% in control and LB silage, respectively) and LAB population (means of 8.14 and 8.59 Log CFU/g in control and LB silage, respectively) found in the present study can be considered high compared with the variations described in the literature for corn silage (3–6% DM) (Kung et al., 2018; Arriola et al., 2021; Agarussi et al., 2022). The high lactic acid concentration could be associated with the high LAB population. At 62 days, fermentation is expected to be stable and the LAB population lower, and these features were observed in the control silage. However, the LAB viability remained stable in the inoculated silage. Heterofermentative LAB, such as L. buchneri, have been reported to survive longer than other LAB in silage, which would explain the larger LAB population in the inoculated silage after storage for 62 days (Carvalho et al., 2017).

The acetic acid concentrations in the present study were within the limits previously reported for corn silage (1–3% DM) (Kung et al., 2018). Inoculation with LB (LBu01) did not increase the acetic acid concentrations over those seen in the uninoculated control silage. A study evaluating eleven strains of L. buchneri showed that some strains produced silages with a lower acetic acid concentration (0.66% DM) than was observed in the control treatment (1.24 DM) (Agarussi et al., 2022). Agarussi et al. (2022) also showed that inoculation with the LB-56.25 strain resulted in silage with a lower acetic acid concentration but greater aerobic stability than in uninoculated control silage. In the present study, the acetic acid concentration and yeast populations did not differ between the inoculated and control silages, but the inoculated silage showed greater aerobic stability. Acetic acid in silage reduces the populations of yeasts and filamentous fungi, which are the main microorganisms associated with aerobic deterioration (Ávila and Carvalho, 2020). Yeasts that can metabolize lactic acid are known to have negative effects on the aerobic stability of silage (Ávila and Carvalho, 2020); therefore, the present results suggest that the yeasts in the inoculated silage had a low capacity to metabolize lactic acid. Thus, the aerobic stability of this silage was lower.

Whole-plant corn silage demonstrated improved aerobic stability after storage for 62 days when inoculated with the L. buchneri LBu01 strain than without inoculation. Although L. buchneri LBu01 did not increase acetic acid concentration, the inoculated silages had lower pH, reduced dry matter loss, higher in vitro digestibility of neutral detergent fiber and dry matter, and improved aerobic stability. These findings indicate that L. buchneri LBu01 can be used in whole-plant corn silage.