Whole-plant corn untreated (Control) or treated with an inoculant (Lentilactobacillus buchneri, Lentilactobacillus hilgardii, and Pediococcus pentosaceus, INO) was ensiled for 210 days in mini-silos at constant (MS-C) or variable temperature (MS-V) or for 220 days in bunkers. Bunker samples were collected at 50 (D50) and 150 (D150) cm below silo surface. Samples from MS-V and MS-C had similar pH and concentrations of lactic acid, propionic acid, 1,2-propanediol, and ethanol, but different acetic acid content (P = 0.009). Additionally, MS-V exhibited greater bacterial (P = 0.002) and fungal (P = 0.011) richness than MS-C. Inoculation decreased (P < 0.001) lactic acid levels while increasing (P < 0.05) acetic acid, 1,2-propanediol, and fungal richness in both mini-silos and bunker. In bunkers, samples collected from D50 had a lower aerobic stability (P < 0.05) than D150, but inoculation increased (P < 0.05) aerobic stability compared to Control, regardless of sampling depth. The storage temperature of mini-silos did not markedly impact the fermentation profile or fungal community. Overall, inoculation increased acetic acid production and fungal diversity in mini-silos, regardless of the storage temperature, and in bunkers, irrespective of sampling the depth, improving the aerobic stability of D50 and D150 bunker silages after long-term ensiling.

The primary objective of haymaking is to dry forage adequately to inhibit the growth of undesirable microbes and halt residual plant enzymatic activity that causes nutrient losses. During the field and storage phases of haymaking, the environment, management practices, and other factors influence the extent of dry matter losses. This review discusses these factors and the strategies that have been developed to mitigate nutrient losses. A major emphasis was placed on hay microbiome dynamics, as it has been scarcely studied despite its importance on nutrient losses during storage and harvest, especially under humid conditions. The effects of cutting height, mower type, and swath manipulation on soil contamination were discussed. Also, the impact of environmental conditions and swath manipulation on wilting time was analysed for humid and arid conditions. Special attention was given to design improvements in harvesting equipment to reduce wilting time and field losses. Furthermore, we assessed the nutrient losses during storage caused by microbial and residual plant enzymatic activity resulting from excessive moisture at baling or re-introduced moisture during storage. The spoilage extent during storage depends on bale moisture, size, density, shape, wrapping, forage type, and storage facilities. A Venn diagram analysis showed that each phase of haymaking process has a unique microbiome and that certain fungal and bacterial genera could be shared across more than one hay production phase. To take corrective actions, hay producers need to be aware of the increased susceptibility to nutrient losses associated with particular field and storage practices, environmental conditions, and forage types.

The effects of farm management practices and seasonal variation on the microbial community and chemical composition of corn and grass-legume silage are largely understudied due to the advantages of controlled mini-silo experiments. This study aims to investigate the effects that some key farm factors (use of an inoculant, farm region, and bunker or tower silo) and seasonal variations have on corn and grass-legume silage from farms across Ontario, Quebec, and New York. The silage was either treated with a commercial inoculant (Lallemand Biotal Buchneri 500® or Chr Hansen SiloSolve FC®) or left untreated. The bacterial communities of silage were compared to those of raw bulk tank milk from the same farm to determine if they were similarly affected by management practices or seasonal variations. Family level analysis of the 16S rRNA V3-V4 gene amplicon bacterial community, the ITS1 amplicon fungal community, NMR water soluble metabolome, and mycotoxin LC–MS were performed on silage over a two-year period. Chemical compounds associated with the use of inoculants in corn and grass-legume silage were higher in inoculated corn (acetate, propane-1,2-diol, γ-aminobutyrate; p < 0.001) and grass-legume (propionate; p = 0.011). However, there was no significant difference in the relative abundance (RA) of Lactobacillaceae in either silage type. Leuconostocaceae was higher in non-inoculated corn (p < 0.001) and grass-legume (p < 0.001) silage than in inoculated silage. Tower silos had higher RA of Leuconostocaceae (p < 0.001) and higher pH (p < 0.001) in corn and grass-legume silage. The one farm that used liquid manure with no other fertilizer type had higher RA of Clostridiaceae (p = 0.045) and other rumen/fecal (p < 0.006) bacteria in grass-legume silage than all other farms. Seasonal variation affected most of the key silage microbial families, however the trends were rarely visible across both years. Few trends in microbial variation could be observed in both silage and bulk tank milk: two farms had higher Moraxellaceae (p < 0.001) in milk and either corn or grass-legume silage. In farms using an inoculant, lower Staphylococcaceae was observed in the raw bulk tank milk.

Spore-forming bacteria in silage may reduce dry matter intake or affect dairy product quality when transferred to milk. The present study investigated the effects of three facultative anaerobes (Bacillus cereus, Bacillus subtilis, and Bacillus licheniformis) and two strict anaerobes (Clostridium tyrobutyricum and Clostridium beijerinckii) commonly found in low-quality silage, milk, and cheese. Maize silage was intentionally contaminated with these spore formers in separate mini silos at 1 × 105 CFU spore former per g and treated with commercial silage inoculant Lentilactobacillus buchneri NCIMB 40788 at 4 × 105 CFU per g or left untreated. The microbial and chemical profiles of maize silage, which were determined using Nuclear Magnetic Resonance (NMR), were measured after fermentation for 100 days, and they were also measured for silage exposed to air for 72 h after opening at 100 days. The effect of the inoculant strain L. buchneri NCIMB 40788 on these contaminated silages was investigated to determine if the inoculant could prevent/limit the negative impacts caused by the spore former contaminants. Overall, inoculation improved silage quality and aerobic stability. Acetic acid content was higher in the INOC samples than in the NIS (p < 0.001), while lactic acid content was lower in INOC than in NIS (p < 0.001). Both lactic and acetic acid levels were higher in the silage samples contaminated with B. cereus. Contamination with the spore formers increased the aerobic and anaerobic spore counts of the samples contaminated with B. subtilis and B. licheniformis compared to the control silage after opening. After 3 days of aerobic exposure, the samples contaminated with B. cereus, B. subtilis, and B. licheniformis showed higher aerobic spore counts than the control. The dominant bacterial population was significantly modified by inoculation. Neither inoculation nor contamination types impacted fungal populations upon opening, but a dominance of Wickerhamomyces was observed after aerobic exposure in the B. cereus silages. The γ-aminobutyrate (GABA) content after aerobic exposure was higher than the uncontaminated control for the silage contaminated with B. licheniformis. The samples contaminated with Clostridium species remained largely unchanged compared to the control samples. Physicochemical and bacterial profiles were mainly affected by inoculation, and a limited impact of spore contaminant was noted. Our results show that when L. buchneri inoculation was carried out, higher aerobic and anaerobic spore counts following contamination with bacterial spore formers were mitigated after reaching aerobic stability.

Inoculants combining Lentilactobacillus buchneri and Lentilactobacillus hilgardii have been shown to improve the aerobic stability of high-moisture corn (HMC) and whole-plant corn silage, but the mode of action of this co-inoculation remains to be elucidated. This study used metatranscriptomics to evaluate the effects of inoculation with L. buchneri alone or combined with L. hilgardii on the bacterial community, gene expression, fermentation profile, and starch digestibility in HMC. High-moisture corn not inoculated (Control) or inoculated with L. buchneri NCIMB 40788 (LB) or L. buchneri NCIMB 40788 combined with L. hilgardii CNCM-I-4785 (Combo) was ensiled in mini silo bags for 30, 60, 120, and 180 days. The fermentation profile was evaluated at all time points. Metatranscriptomics was performed on samples collected on day 120. Combo had a greater alpha diversity richness index of contigs than LB and Control, and inoculation with Combo and LB modified the beta-diversity of contigs compared to Control. Out of 69 genes of interest, 20 were differentially expressed in LB compared to Control and 25 in Combo compared to Control. Of those differently expressed genes, 16 (10 of which were associated with carbohydrate metabolism and six with amino acid metabolism) were differently expressed in both LB and Combo compared to Control, and all those genes were upregulated in the inoculated silages. When we compared Combo and LB, we found seven genes expressed differently, four associated with carbohydrate metabolism and downregulated in Combo, and three associated with amino acid metabolism and upregulated in Combo. At day 120, the inoculated silages had more culturable lactic acid bacteria, higher Lactobacillus relative abundance, and lower Leuconostoc relative abundance than Control. The concentration of acetic acid remained low throughout ensiling in Control, but in LB and Combo, it increased up to day 60 and remained stable from day 60 to 180. The 1,2-propanediol was only detected in LB and Combo. Inoculation did not affect the concentration of starch, but starch digestibility was greater in Combo than in Control. Inoculation of HMC with Combo modified the gene expression and fermentation profile compared to Control and LB, improving starch digestibility compared to uninoculated HMC.

Triticale, a small-grain forage, was harvested for an ensiling experiment evaluating different silage inoculants. Fresh material (mean dry matter 404.1 g kg−1) was wilted, chopped, and assigned to one of four treatment groups including water only (Control), heterofermentative strains Lentilactobacillus buchneri (LB), Lentilactobacillus hilgardii (LH), or combination (Combo) of both. Inoculants were applied at 4.0 x 105 CFU per g of fresh forage, and the Combo contained both strains at 2.0 x 105 CFU per g. Treated forage was packed into 7.57 L mini-silos for openings after 15, 30, and 130 d. Samples were collected at each opening for microbial enumeration of lactic acid bacteria, enterobacteria, yeasts, and moulds. Additional samples were collected for analysis of fermentation profiles, and nutritional analysis following dry matter determination and grinding. Aerobic stability was also evaluated at each opening through a 10-d period. Fermentation profiles were notably affected, including increases in acetic acid (g/kg DM) in LB and Combo treated silage after 15 d which resulted in reduced pH. Aerobic stability was vastly improved through inoculation by prolonging the time to reach 2˚ C above ambient, which was likely a result of decreased yeast counts. Our research validates the use of combined heterofermentative lactic acid bacteria strains on an ensiled small-grain specie, and further agrees with previous findings of prolonged aerobic stability through increases in lactate and acetate in response to co-inoculation.

Aims This study evaluated changes in epiphytic microbial population of alfalfa (Medicago sativa) during the growing season. First cut forage was harvested to study the effects of an inoculant combining two obligate heterofermentative lactic acid bacteria strains on the bacterial and fungal communities and the fermentation of alfalfa silage. Methods and Results The epiphytic microbiome of alfalfa was evaluated 10-times during the growing season. Alfalfa wilted to 395.0 g/kg was treated with water (Control) or with a combination of L. buchneri NCIMB 40788 and L. hilgardii CNCM-I-4785 (LBLH). Mini-silos were opened after 1, 4, 8, 16, 32, and 64 days of ensiling. The relative abundance (RA) of the epiphytic bacterial and fungal families varied during the growing season. After 1 day, Weissella was the most abundant genus and present at similar RA in the two treatments (average 80.4%). Compared with Control, LBLH had a higher RA of Lactobacillus at day 1, 16, 32, and 64, and a lower RA of Weissella from day 8 to 64. Control contained more bacteria belonging to the Enterobacteriales than LBLH up to day 16. Inoculated silage had more acetate than Control at day 32 and 64. The fungal population were similar between treatments. The enhanced development and dominance of Lactobacillus in inoculated silage led to greater accumulation of acetate and propionate, which reduced the numbers of culturable yeasts but did not markedly affect the fungal community structure. Conclusions The bacterial community composition of alfalfa stands in the filed changed over time and was affected by cutting. For the ensiling trial, inoculation modified the composition of the bacterial community of alfalfa, increasing the RA of Lactobacillus while reducing the RA of Weissella and of Enterobacteriaceae. Significance and Impact of Study Inoculation increased the RA of Lactobacillus, hampering the dominance of Weissella in the early stages of ensiling, improving antifungal compounds production and reducing the numbers of culturable yeasts.

This study evaluated the effects of novel silage inoculants containing lactic acid bacteria (LAB) and fibrolytic enzymes on ensiling, aerobic stability (AS), and the performance of growing beef cattle. Whole-plant corn forage was either uninoculated (CON) or inoculated with a mixture of LAB containing (cfu g-1 fresh forage) 1.5 × 105 L. hilgardii (CNCM I-4785), 1.5 × 105 L. buchneri (NCIMB 40788) and 1.0 × 105 P. pentosaceus (NCIMB 12455) for a total of 4.0 × 105 cfu g-1 fresh forage LAB (IB), or a combination of IB plus fibrolytic enzymes (xylanase + β-glucanase) (IC). All treatments were ensiled in mini-silos, whereas CON and IC were also ensiled in silo bags for the growth performance study. Total bacteria (TB) counts were lower (P = 0.02) for IC than CON after 14 d of ensiling, whereas TB counts of IC and IB were greater (P ≤ 0.01) than CON after 60 d of ensiling in mini-silos. The LAB in IC and IB ensiled in mini-silos were greater than CON on d 60 (P ≤ 0.01) and 90 (P ≤ 0.001) of ensiling and after 3 d (P ≤ 0.01) of aerobic exposure (AE). Silage pH of IC ensiled in silo bags was lower than CON on d 3 (P < 0.01), 7 (P < 0.001), and 14 (P = 0.02) of AE. Yeast counts were lower for IC than CON in terminal silage (P < 0.001), and after 3 (P < 0.001) and 7 d (P < 0.01) of AE. Acetate (AC) concentrations were higher (P ≤ 0.02) for IC than CON throughout AE, whereas lactate (LA) concentrations of IC were greater than CON on d 3 (P < 0.001), 7 (P < 0.01), and 14 (P < 0.001) of AE. Greater AC concentration and lower yeast counts resulted in greater (P < 0.001) stability for IC ensiled in silo bags than CON after 14 d of AE. Growth performance of steers was similar (P > 0.05) as the nutrient composition of silage was similar across diets. Improved AS of IC could potentially have a greater impact on DMI, production efficiency, and growth performance in large-scale commercial feedlot operations where silage at the silo face may be exposed to air for longer periods of time.

Zearalenone (ZEA) is a mycotoxin widely occurring in many agricultural commodities. In this study, a purified bacterial isolate, Bacillus sp. S62-W, obtained from one of 104 corn silage samples from various silos located in the United States, exhibited activity to transform the mycotoxin ZEA. A novel microbial transformation product, ZEA-14-phosphate, was detected, purified, and identified by HPLC, LC-MS, and NMR analyses. The isolate has been identified as belonging to the genus Bacillus according to phylogenetic analysis of the 16S rRNA gene and whole genome alignments. The isolate showed high efficacy in transforming ZEA to ZEA-14-phosphate (100% transformation within 24 h) and possessed advantages of acid tolerance (work at pH = 4.0), working under a broad range of temperatures (22–42 °C), and a capability of transforming ZEA at high concentrations (up to 200 µg/mL). In addition, 23 Bacillus strains of various species were tested for their ZEA phosphorylation activity. Thirteen of the Bacillus strains showed phosphorylation functionality at an efficacy of between 20.3% and 99.4% after 24 h incubation, suggesting the metabolism pathway is widely conserved in Bacillus spp. This study established a new transformation system for potential application of controlling ZEA although the metabolism and toxicity of ZEA-14-phosphate requires further investigation.

Abstract Aerobic deterioration of silage following feeding out is responsible for the deterioration of its quality. Inoculation of silage with lactic acid bacteria is one strategy to limit these effects. A trial was performed using whole‐plant corn ensiled in bag silo, and forage was inoculated with Lentilactobacillus buchneri NCIMB 40788 (Lactobacillus buchneri) and Lentilactobacillus hilgardii CNCM‐I‐4785 (Lactobacillus hilgardii) or not (Control silage). After 159 days of fermentation, the silos were opened and the silage was sampled at 24‐h intervals during a 10‐day aerobic stability assay to measure pH, the fermentation profile, mycotoxins, and microbial and fungal populations. In inoculated silage, lactic acid concentrations and pH remained stable during the aerobic phase and higher microorganism alpha‐diversity was observed. Treated silage was characterized by a high abundance of Saccharomycetes and maintenance of Lactobacillus throughout the aerobic stability assay. The high aerobic stability of the inoculated silage contrasted with the decrease in lactic acid contents and the increase in pH observed in the Control silage, concomitantly with an increase in lactate‐assimilating yeast (Pichia and Issatchenkia), and in Acetobacter and Paenibacillus OTUs. Remarkably, Penicillium and roquefortine C were detected in this silage by day 8 following exposure to air. Our study highlighted the fact that the use of L. buchneri with L. hilgardii modified the consequences of exposure to air by maintaining higher microbial diversity, avoiding the dominance of a few bacteria, and preventing fungi from having a detrimental effect on silage quality.

Two separate experiments were conducted to compare the nutrient digestion and growth performance of beef cattle fed diets containing up to 77% of two conserved alfalfa hays. The two alfalfa hays were baled either at the optimum moisture (8.6%) or at a higher-than optimum moisture (21.4%) with application of a mixture of Pediococcus pentosaceus and chitinase at baling. Digestibility of dry matter, neutral detergent fibre, acid detergent fibre, rumen parameters, and growth performance were similar (P ≥ 0.16) for cattle fed either diet. A combination of P. pentosaceus and chitinase has the potential to preserve high-moisture alfalfa hay.

Aims To evaluate the capacity of Lactobacillus hilgardii and Lactobacillus buchneri on modifying the bacterial community and improving fermentation and aerobic stability of high-moisture corn (HMC). Methods and Results High-moisture corn was untreated (CTR), treated with L. hilgardii (LH) or L. buchneri (LB) at 600 000 CFU per gram fresh weight, or with L. hilgardii and L. buchneri at 300 000 CFU per gram fresh weight each (LHLB), and stored for 10, 30 or 92 days. Compared to CTR, inoculated silages had higher Lactobacillaceae relative abundance, lower yeasts numbers and higher aerobic stability. Treatment with LHLB resulted in a higher acetic acid concentration than LH and higher 1,2 propanediol concentration than LB, such differences were numerically greater at 10 and 30 days but statistically greater at 92 days. At 10 days, all inoculated silages were more stable than CTR, but LHLB was even more stable than LB or LH. Conclusions The combination of L. hilgardii and L. buchneri had a synergistic effect on yeast inhibition, leading to greater improvements in aerobic stability as early as 10 days after ensiling. Significance and Impact of the Study Lactobacillus hilgardii, especially in combination with L. buchneri, can improve the aerobic stability of HMC after a very short period of ensiling.

This study evaluated the effects of inoculation of whole crop corn silage with a mixture of heterofermentative lactic acid bacteria (LAB) composed of Lactobacillus hilgardii and Lactobacillus buchneri on ensiling, aerobic stability, ruminal fermentation, total tract nutrient digestibility, and growth performance of beef cattle. Uninoculated control corn silage (CON) and silage inoculated with 3.0 x 10(5) cfu g(-1) of LAB containing 1.5 x 10(5) cfu g(-1) of L. hilgardii CNCM 1-4785 and 1.5 x 10(5) cfu g(-1) of L. buchneri NCIMB 40788 (INOC) were ensiled in silo bags. The pH did not differ (P > 0.05) between the two silages during ensiling but was greater (P < 0.001) for CON than INOC after 14 d of aerobic exposure (AE). Neutral detergent insoluble crude protein (NDICP) content (% of DM and % of CP basis) of terminal INOC silage was greater (P <= 0.05) than that of CON. In terminal silage, concentrations of total VFA and acetate were greater (P < 0.001), while water-soluble carbohydrates were lower (P < 0.001) for INOC than CON. Yeast and mold counts were lower for INOC than CON (P <= 0.001) in both terminal and aerobically exposed silages. The stability of INOC was greater (P < 0.001) than that of CON after 14 d of AE. Ruminal fermentation parameters and DMI did not differ (P > 0.05) between heifers fed the two silages, while there was a tendency (P <= 0.07) for lower CP and starch digestibility for heifers fed INOC than CON. Total nitrogen (N) intake and N retention were lower (P <= 0.04) for heifers fed INOC than CON. Dry matter intake as a percentage of BW was lower (P < 0.04) and there was a tendency for improved feed efficieny (G:F; P = 0.07) in steers fed INOC vs. CON silage. The NEm and NEg contents were greater for INOC than CON diets. Results indicate that inoculation with a mixture of L. hilgardii and L. buchneri improved the aerobic stability of corn silage. Improvements in G:F of growing steers fed INOC silage even though the total tract digestibility of CP and starch tended to be lower for heifers fed INOC are likely because the difference in BW and growth requirements of these animals impacted the growth performance and nutrient utilization and a greater proportion of NDICP in INOC than CON.

Silage making is not a novel technique. However, the agricultural industry has made great strides in improving our understanding of—and efficiency in—producing high-quality silage for livestock. Silage microbiology research has been using the newest molecular techniques to study microbial diversity and metabolic changes. This chapter reviews important research that has laid the foundation for field-based utilization of silage inoculants. We also outline areas of current, and future, research that will improve global livestock production through the use of silage.

Plate counts using selective culture media is still the most frequently used method for the enumeration of the different microbial groups that colonize silage, including lactic acid bacteria, yeasts and molds. Since different culture media have specific composition, they may allow the growth of specific populations. To date, no study has used next generation sequencing technology to compare the selective capacity of these different culture media although this approach could provide comprehensive insight into the relevance of using one culture medium over another. Sequencing of the 16S rDNA and ITS amplicon were performed to compare the selectivity of different culture media used in silage microbiology. Corn silage, grass-alfalfa silage and total mixed ration extracts were plated on five selective media for lactic acid bacteria, incubated under aerobic and anaerobic conditions, and on eight selective media for yeast and molds to compare their selectivity. Ensiling provided a pre-selection environment for specific microorganisms over forage and reduced the number of observed OTUs: only 12 OTUs of bacteria were observed in corn silage sampled in the center of a bunker silo, while the mean number of OTUs identified in samples taken closer to the side of the silo, influenced by higher oxygen and humidity level, increased to 79. Still, MRS and Rogosa plates had less than 12 different OTUs in the center and 24 at the side, mainly Lactobacillaceae, Acetobacteraceae, and Leuconostocaceae. Incubating the plates under anaerobic conditions was selective against Acetobacteraceae. MRS supplemented with acetic acid increased selectivity of lactic acid bacteria. When plated on culture media specific for yeast and molds, from 17 to 68 different OTUs were observed in corn silage. Mixed grass-alfalfa silage and total mixed ration samples usually had more observed OTUs and the diversity profile of the corresponding culture media was similar to that of the original samples. For yeasts and molds, Dichloran Rose Bengal Chloramphenicol Agar revealed a diversity profile close to the that of the corn silage.

Objective:To evaluate the effects on microbial diversity and biochemical parameters of gradually increasing temperatures, from 5 to 25 °C on corn silage which was previously fermented at ambient or low temperature. Methods:Whole-plant corn silage was fermented in vacuum bag mini-silos at either 10 or 20 °C for two months and stored at 5 °C for two months. The mini-silos were then subjected to additional incubation from 5 to 25 °C in 5 °C increments. Bacterial and fungal diversity was assessed by PCR-DGGE profiling and biochemical analysis from mini-silos collected at each temperature. Results:A temperature of 10 °C during fermentation restricted silage fermentation compared to fermentation temperature of 20 °C. As storage temperature increased from 5 to 25 °C, little changes occurred in silages fermented at 20 °C, in terms of most biochemical parameters as well as bacterial and fungal populations. However, a fairly high number of enterobacteria and yeasts (4-5 log10 CFU g FM-1) were detected at 15 °C and above. PCR-DGGE profile showed that Candida humilis predominated the fungi flora. For silage fermented at 10 °C, no significant changes were observed in most silage characteristics when temperature was increased from 5 to 20 °C. However, above 20 °C, silage fermentation resumed as observed from the significantly increased number of LAB colonies, acetic acid content, and the rapid decline in pH and WSC concentration. DGGE results showed that Lactobacillus buchneri started to dominate the bacterial flora as temperature increased from 20 to 25 °C. Conclusion:Temperature during fermentation as well as temperature during storage modulates microorganism population development and fermentation patterns. Silage fermented at 20 °C indicated that these silages should have lower aerobic stability at opening because of better survival of yeasts and enterobacteria.

Lactic acid bacteria (LAB) used as silage additives have been shown to improve several fermentation parameters, including aerobic stability. Inoculation with a combination of Lactobacillus buchneri NCIMB40788 and Lactobacillus hilgardii CNCM-I-4785, contributes to an increase in aerobic stability, compared to each strain inoculated independently. To understand the mode of action of the combination on the LAB community, a fermentation-kinetic study was performed on corn. Four treatments, Control, Lb. buchneri, Lb. hilgardii, and a combination of the two strains, were fermented 1, 2, 4, 8, 16, 32, and 64 days. Corn silage inoculated by both strains had a lactate:acetate ratio of 0.59 after 64 days and a higher concentration of lactate than Lb. buchneri. Analysis of the microbiota by 16S and ITS amplicon metasequencing demonstrated that inoculation led to lower bacterial diversity after 1 day, from 129.4 down to 40.7 observed operational taxonomic units (OTUs). Leuconostocaceae represented the dominant population by day 1, with 48.1%. Lactobacillaceae dominated the succession by day 4, with 21.9%. After 32 days, inoculation by both strains had the lowest bacterial alpha diversity level, with 29.0 observed OTUs, compared to 61.3 for the Control. These results confirm the increased fermentation efficiency when the two Lactobacillus strains are co-inoculated, which also led to a specific yeast OTUs diversity profile, with Hannaella as the main OTU.