A study of fermentation characteristics, nutrient content, and microbial population of triticale silage produced with different lactic acid bacteria: Long-term preserved triticale silage for livestock

1. Introduction

It has become increasingly important to develop high quality silage from grasses and legumes with enriched fermentative metabolites because animal farming has increased dramatically. Triticale is a hybrid variety of wheat and rye that produces more biomass and CP than wheat alone, making it useful in livestock animal feed production. The chemical composition of triticale is closer to wheat than rye; so, it could be used as a food source for humans and animals. In recent years, triticale has been considered good forage for making high-quality silage for livestock due to its high CP content (Harper et al., 2017, Soundharrajan et al., 2019, Lin et al., 2021, Jung et al., 2022, Negi et al., 2022). It is well known that microbial additives have gained considerable attention for the purpose of achieving a controlled fermentation of plant-based silages and increasing their digestibility, as evidenced by the fact that inoculants have been developed as silage additives for over four decades (Okoye et al., 2023). LA bacteria have been extensively studied for their potential to improve human and animal health (Amaral et al., 2020). Furthermore, LAB is widely regarded as an efficient and reliable method of storing forage in animal production (Guo et al., 2023). Researchers have found that understanding biological transitions in the ensiling process has helped them focus on unique strains that are becoming more efficient during silage ensiling (Xu et al., 2019). Due to their high safety and feasibility, non-corrosiveness, eco-friendliness, improved dry matter (DM) recovery, fermentation characteristics, and animal performance, LAB is considered to be a more suitable inoculum for silage production than the other additives (Okoye et al., 2023). LAB produces LA, AA, BA, succinic acid, and other organic acids during ensiling. Silage acidification inhibits microbial growth and prevents aerobic degradation (Muck 2010, Muck et al., 2018). Animal health and food quality can also be affected by spoilage microorganisms. LAB has been used for many years to improve the silage quality at LM and HM conditions and to increase milk production, body weight, and feed efficiency (Muck et al., 2018). Addition of LAB additives to crop biomass during ensiling to increase the positive fermentation, reduce DM loss, reduction of aerobic deterioration during feed out, improve the hygienic quality of the silage and aerobic stability restrict or limit the secondary fermentation via inhibition of undesirable microbes, increase the nutritive values of the silage, and mitigate the methane emission (Kaewpila et al., 2021, Ridwan et al., 2023).

Lactobacillus sp, pediococcus sp, and enterococcus sp are the major genera involved in fermenting silage anaerobically. Inoculants used intensively for silage production are Lactobacillus plantarum, Lactobacillus brevis, Pentosaceus pentosaceus, Lactobacillus rhamnosus, and Lentilactobacillus bucbneri, as well as Enterococcus faecium (Ogunade et al., 2019, Puntillo et al., 2020, Jung et al., 2022). Ensiled forage material deteriorates in the presence of oxygen when the silo is opened, particularly lactate-assimilating yeast. When silage temperature and pH rise, undesirable microbial growth occurs. In order to address this issue, LA bacteria have been inoculated into silage for many decades (Filya 2003, Kung et al., 2018). Direct ensiling is difficult due to the limited number of LAB in native plants. In recent years, new LABs have been isolated to produce silage, yet this activity remains important worldwide (Paradhipta et al., 2020, Dos Santos Leandro et al., 2021), since more strains are being sought not only as silage inoculants, but also for other plant-based foods for humans and animals (Wuyts et al., 2020). LAB strains promote silage fermentation as a single culture in most studies. It is interesting to note that silage produced with LAB in mixed cultures (more than one strain) accelerated its acidification, enhanced its LA production, and reduced BA by its synergistic effects, compared with silage produced with single LAB (Filya 2003, Zhang et al., 2023). Considering these factors, we examined different types of LA bacteria such as Pediococcus pentosaceus (2), Lactobacillus plantarum (1), and Lactobacillus rhamnosus (1) effects on acidification, fermentative metabolites, microbial population, and nutrient levels in triticale silage at the different moisture conditions after 6 and 12 months. In addition, a triangle map with a correlation matrix was used to determine the interactions among microorganisms, organic acids, and nutrient contents of the experimental silages.

2. Materials and methods

3. Results

3.5 Triangle heatmap correlation between fermentative parameters, nutrient contents, and microbial population

An interaction among pH, organic acids, nutrient content, and microbes was investigated using Python software. The pH of the silages was negatively correlated with LA, AA, and LAB populations. In contrast, yeast and mold were positively correlated with pH and negatively interacting with AA in the silages after 6 months in both HM (Figure 1a) and LM (Figure 1b) silages. Lactate content of the silages was positively correlated with LAB, and negatively correlated with BA. After 12 months’ storage period, similar patterns of interactions were noted among pH, LAB, LA, AA, yeast, and mold in both HM (Figure 1c) and LM (Figure 1d) silages.

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Figure 1.

Triangle heatmap-interactions among fermentative acids, nutrient contents, and microbial population by Python software. (a) Six month fermented silage at HM condition, (b) six month fermented silage at LM condition, (c) twelve-month fermented silage at HM condition, and (d) twelve-month fermented silage at LM condition. LA- Lactic acid, AA-Acetic acid, BA-Butyric acid, ADF-Acid detergent fiber, NDF- Neutral detergent fiber, CP-Crude protein, LAB- Lactic acid bacteria.

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4. Discussion

Forage conservation by ensiling has gained considerable attention for providing reliable and predictable feed for ruminants. A number of factors, such as plant oxidation, pathogens in plants, proteolytic activity, Clostridia fermentation, and microbial deamination and decarboxylation of amino acids, can be detrimental forage productivity, which increases the accumulation of anti-nutritional complexes in forage samples (Oliveira et al., 2017). Several decades, LA bacteria have been used to promote the fermentation process in plant samples for livestock due to its efficiency to improve the quality of the silages via acidification by production of LA at higher levels and reduced BA content and inhibition of undesirable microbial growth which favor the long-term storage of fermented silages (Feng et al., 2023, Guo et al., 2023). In spite of LAB’s benefits, maintaining quality of silage during long-term storage has several challenges which includes aerobic stability, temperature fluctuations, and moisture content, which may lead to spoilage. Furthermore, improper sealing and handling can intensify these issues, resulting in lower feed quality and higher economic losses.

In addition, epiphytic LAB in the plants has been varied from 10¹ to10⁷ CFU/g based on several environmental factors (Pahlow et al., 2003) such as DM content of samples, temperature, and storage period. Epiphytic LAB in plants is not sufficient to induce rapid fermentation in ensiled silages. To produce excellent quality silage with high digestibility, different flavors, and additional inoculum must be incorporated into the ensiling method. LAB is used as an inoculant to increase the proportion of LA to AA in silage making to inhibit proteolysis and increase DM recovery (Muck 2013, Kim et al., 2017).

Numerous studies reported that adding LAB during the ensiling of different forages can accelerate positive fermentation (Kim et al., 2017, Guo et al., 2023). In addition, the production of high-quality silage with mixed forms of LAB (more than one strain) could accelerate the fermentation process due to its synergistic functions to enhance LA production and reduce BA levels compared to silage produced with single LAB treatments. Furthermore, mixed LAB could extend storage periods with preserved nutrients (Filya 2003, Kleinschmit and Kung 2006, Muthusamy et al., 2021, Zhang et al., 2023). In the present study, high-quality silage was made from heading stages triticale forage at different moistures with various LA bacteria such as L. plantarum, P.pediococcus, and L. rhamnosus in either single or cocktail form and stored for 6 / 12 months. After storage periods, the fermentative parameters such as nutrient and microbial profiles were evaluated. High-quality silage has always been enriched with nutrients that preserve fermentation potential. ADF and NDF are key indicators of silage quality (He et al., 2018, Li et al., 2018, Xian et al., 2022). Fermented samples with increased ADF and NDF content indicate poor quality silage and reduced animal digestion. Silage with high fiber contains lower protein and energy than lower fiber silage. Hence, reducing the fiber content of silages is a better strategy to improve feed value (Li et al., 2008). Our study found that silage produced with different types of LAB did not change nutrient contents such as ADF, NDF, and CP. But, in our previous study, silage produced with L. plantarum KCC - 48 and mixed LAB such as L. plantarum, P. pentosaceus, and L. rhamnosus combination significantly decreased ADF of HM early heading triticale silages after 180 and 360 days’ fermentation (Soundharrajan et al., 2023). The same strains were used in the present study to ferment heading stage of triticale. However, there is no response to degradation of fiber material in fermented silage after 6 and 12 months. It may be richer and stronger fibers in the heading stage of triticale than in the early heading stage, which is the reason for being unable to degrade by the currently used LAB.

Acidification is a key indicator for production of high quality silages which reduces proteolytic activities in silages, decomposition of nutrients, and inhibits undesirable microbial growth (Muck 2010). An ideal pH for the good fermentation of silages ranges between 3.8 and 4.2 pH (Ahmadi et al., 2019). In general, pH 4.2 is considered a benchmark for well-fermented silages (He et al., 2018). In the present study pH of the non-inoculated silages had higher pH values greater than five in both HM and LM condition after 6- and 12-month storage periods. It is due to lower LAB population and higher yeast and mold counts in non-inoculated silages, as evidenced by yeast and mold were positively correlated with pH of the silages determined by heatmap correlation. Silage produced with different LAB either single or cocktail form strongly reduced the pH of silage at both moisture and storage conditions due to higher LAB and lower yeast and mold populations. The previous supportive evidence for LAB strains actively suppressed fungal growth by pH 4.0 and the presence of LA (Broberg et al., 2007). The pH of the LAB mediated silages ranged from 3.98 to 4.47 and 3.94 to 4.49 in both HM and LM silage after 6 and 12 months’ fermentation, respectively. At both moisture levels and storage periods, the pH almost reached desirable levels in response to different LAB treatments. In particular, cocktail-I and cocktail-II treatments more strongly reduced the pH of the silage than single strain treatments. Even after 12 months, the pH of the silage made with cocktail maintained the pH below 4.2 benchmark level, it was consistent with heatmap correlation study, suggesting that the higher LAB in treatment was negatively correlated with pH and negatively correlated with yeast and mold after 6 and 12 months at both moistures.

A high-LAB population and a low yeast and mold count were achieved by adding LA bacteria in different forms to the silages. In order to confirm the fermentation quality of forage samples, it is important to measure the LA content, which is an essential acid and an indicator of a successful fermentation process. LA is a predominant acids present in the fermented silages than the other acids (Wang et al., 2021). It is more than 10-12 times higher than other organic acids that contribute to reducing the pH of silage (Kung et al., 2018). In both HM and LM conditions, non-inoculum treated silage had lower levels of LA after 6 and 12 months. This may be due to a low concentration of LAB was in non-inoculum treated silages, confirming that the native LAB present in the plants cannot induce LA fermentation. When silage is made with different LAB strains, either in single or cocktail forms, the amount of LA produced is more than 7 fold higher than non-inoculum treated silage. The production of LA after 6 months in HM silage made with cocktail-I or cocktail-II was more than 10 times compared to non-inoculum treated silage. In LM silage at 6 months, LA was not detected in non-inoculum treated silages. But, silage treated with LAB at the different forms produced LA ranging from 2.04 ± 0.05 to 4.34 ± 0.19 DM%, it confirmed that addition of LAB produced several folds more LA than non-inoculum treated silage. Similar trends were observed in silage treated with LAB after 12 months of storage periods. Higher production of LA in silages due to higher population of LAB and lower counts of yeast and mold. The correlation study confirmed that LA was positively correlated with LAB and negatively correlated with pH, yeast and mold counts, suggesting that LAB plays a main role in pH reduction and higher LA production. Subsequently, AA is another most leading acid in fermented silage (Hafner et al., 2013, Kung et al., 2018). Marginal level of AA has been accepted for good quality silage which inhibits yeast and mold growth and increases aerobic stability (Wilkinson and Davies 2013). The acceptable range of AA is from 10g/kgDM to 30g/KgDM (Muck et al., 2018, Gerlach et al., 2021). Triticale silage produced at heading stage with LAB in single or cocktail form significantly increased the content of AA compared to non-inoculum silages. The production of AA in LAB-treated silages varies among the strains used. KCC-45, KCC-48, cocktail-I, and cocktail-II produced higher AA acid than other strains (p<0.05) at HM conditions whereas all of the used LAB in any forms increased AA content after 6 months in LM. After 12 month of storage, KCC-45 and KCC-48 at HM and KCC-54 and cocktail-I in LM conditions significantly increased AA levels compared to non-inoculum silages. The production of AA at marginal levels could be beneficial for silage due to inhibition of yeast and mold, as evidence that the triangle heatmap correlation revealed that AA was negative correlation with yeast and mold growth at all moisture and storage periods.

BA indicates poor quality silage due to clostridia-mediated fermentation (Pahlow et al., 2003, Zhang et al., 2023). Reduction or inhibition in total is a great strategy for producing high-quality silage with preserved nutrients for long-term storage. Since soluble nutrients have been degraded by clostridia fermentation, higher levels of BA reduce of silage DM and nutritional content (Kung et al., 2018). The study found that HM silage at 6 months had higher concentrations of BA in control silage which were reduced by LAB treatments in different forms, such as KCC-45, KCC-48, KCC-54, and both cocktail forms (p <0.05). Despite this, no significant amount of BA is produced in all experimental silages at LM levels. After 12 months of storage, LAB treatments significantly reduced BA levels in both high and low silages. Variations in BA reduction were observed among strains. Cocktail-I and cocktail-II treatments significantly reduced BA production compared to LAB alone. It is believed that the reduction in BA levels in LAB-treated silage is due to the inhibition of yeast and mold, and to LAB dominance. In addition, BA showed a positive correlation with yeast and mold, while LAB showed a negative correlation with BA. There are specific challenges in producing triticale silage at heading stages due to a lack of water soluble carbohydrates (WSCs). The heading stage triticale contains less WSC and more fiber (ADF and NDF) than the early heading stage (milky stage). WSC is essential to LAB fermentation since it increases LA levels which reduce pH. Generally, fiber content is not favorable for LAB-mediated fermentation. Thus, the use of LAB that produces cellulase during the ensiling of fiber-rich forages could enhance fermentation and increase essential organic acids content.

5. Conclusions

The production of high-quality silage from plant sources is essential for livestock farms. However, storing silage for a long period of time can lead to mold and yeast growth, as well as LA degradation. Thus, biological additives, such as LA bacteria, improve fermentation quality, and preserve nutrients for long-term storage. Therefore, high-quality silage was made from heading triticale with different types of LA, such as Pediococcus pentosaceus, Lactobacillus plantarum, and Lactobacillus rhamnosus, by the ensiling method to speed up fermentation by reducing pH of silages and periods after six and twelve months. LAB treatments also retained more LA and reduced BA levels by increasing LAB and reducing yeast and mold counts even after 12 months of storage. Cocktail-I (two P. pentosaceus and one L. plantarum) and cocktail-II (L. plantarum, P. pentosaceus and L. rhamnosus) treatments were significantly more effective at preserving nutrient values of fermented silages for livestock for long periods of time than single strain treatments. This suggests that silage developed using multiple strains as a cocktail is more appropriate for livestock for long-time storage. Despite this, further studies are required to confirm the efficiency of the different LAB used in this study in either single or cocktail form on fermentation of different forages, including grasses and leguminous plants.