Oat (Avena sativa L.) is a crucial annual crop with extensive application as forage crop across the world. In China, oats are primarily cultivated in regions with cool climates. Ensiling is a method of preserving forage crops through fermentation, which produces acids that help in maintaining the forage. However, low temperatures during the harvest stage can reduce the microbial activity, hinder the fermentation process (Zhou et al., 2016; Li et al., 2021), leading to inadequate acid production in silage (Cao et al., 2011; Zhang et al., 2021). This results in poor preservation of the oat forage, which may negatively affect the nutritional quality and safety of the silage. Despite the importance of ensuring high-quality silage, current research has largely overlooked the challenges associated with fermentation in cold environments, especially in regions with naturally low temperatures. Although bacterial inoculants are commonly used to enhance fermentation, Bernardes et al. (2018) pointed out that such bacterial inoculants may be less effective at low temperatures, as most silage inoculants are developed from bacterial strains that thrive in warmer conditions (Weinberg and Muck, 1996). This raises a critical issue in silage production in colder climates—existing additives and inoculants are often not suited for low-temperature conditions. Thus, it is essential to explore new methods and develop preparation techniques that can enhance fermentation quality at colder environment. Chemical additives such as formic acid (FA), propionic acid (PA) and sodium benzoate (SB) are effective in controlling spoilage and pathogenic microorganisms. While the majority of studies have focused on the effects of these chemical additives on silage fermentation at moderate temperatures, research on silage under low-temperature conditions remains limited, particularly with respect to biogenic amine formation. However, investigating their impact at low temperature is equally important, particularly for regions with colder climates or for situations where oat silage might be stored in less-than-ideal conditions.

Biogenic amines are low molecular weight nitrogenous organic compounds formed in silage through the decarboxylation of amino acids (Liang et al., 2024). This process is primarily facilitated by microorganisms during the fermentation of silage. Some biogenic amines such as putrescine, cadaverine, tyramine, histamine, and spermidine are commonly found in silage and can significantly impact its quality and safety (Scherer et al., 2015). The presence and concentration of these amines varies depending on factors such as the forage types, ensiling conditions, and the microbial populations present during fermentation process. An appropriate level of biogenic amines is essential for normal cell growth and differentiation, excessive amounts can adversely affect feed intake and livestock health (Driehuis et al., 2018). High levels of putrescine can decrease nitrogen degradability in the rumen of steers, which can impair protein utilization (Dawson and Mayne, 1997). Tyramine can raise the pH and increase the proportion of isovalerate in rumen fluid, which can affect rumen fermentation dynamics and animal performance (Dawson and Mayne, 1996). Significant levels of biogenic amines have been observed in silages made from perennial ryegrass, maize, cocksfoot, alfalfa and clovers (Van Os et al., 1996; Nishino et al., 2007; Skladanka et al., 2017). Temperature plays a crucial role in the formation of biogenic amines in fermented food (Ormanci and Colakoglu, 2017), with Peñas et al. (2010) specifically noted that low temperature can lead to the accumulation of biogenic amines due to slower acidification and altered microbial activity. Few studies have explored the role of low-temperature-resistant lactic acid bacteria (LAB) and the effectiveness of chemical additives in mitigating biogenic amine formation in low-temperature environment.

In the present study, we investigate the potential of low-temperature-resistant LAB and chemical additives (PA, FA, and SB) to improve silage quality during fermentation in cold conditions. Moreover, the possibility of decreasing the concentrations of biogenic amines during low-temperature fermentation with LAB and chemical additives was tested.

Low temperature restricts silage fermentation (Li et al., 2021), while extensive studies have been conducted on silage at moderate and high temperatures, research on silage at low temperature is limited. This study explored methods to improve oat silage quality under low-temperature conditions. We isolated the low-temperature-resistant strain Y28 and applied it, along with chemical additives propionic acid (PA), formic acid (FA) and sodium benzoate (SB), to oat silage to enhance its quality at low-temperature. Additionally, the production of biogenic amines during low-temperature ensiling was investigated.

The isolated strain Y28 exhibited robust growth capabilities at low temperature (10°C) and low pH (3.5), demonstrating psychrotolerance and acid tolerance. Inoculating oat forage with strain Y28 during ensiling notably improved the fermentation quality of oat silage under low-temperature conditions. The application of Y28 significantly reduced the presence of putrescine, cadaverine, histamine, tyramine, spermidine and total biogenic amines in silage. Amino acid decarboxylase activity assays revealed that strain Y28 has the capability to produce putrescine in synthetic screening media containing precursor amino acids. However, this potential does not necessarily translate to putrescine production under the complex conditions of ensiling, as confirmed by our experiments. It suggests that the impact of LAB species like Y28 on biogenic amine production may not solely stem from their own enzymatic activities, but also from their broader effects on fermentation quality. Min et al. (2007) reported that the organic acid, such as lactic acid, can reduce biogenic amines in meat. In this study, Y28 significantly increased lactic acid production, which could potentially inhibit the growth of pathogenic microorganisms and thereby prevent excessive accumulation of biogenic amines in silage. There is limited literature on the effects of different additives on the concentrations of individual and total biogenic amine in silage at low temperature. In fresh oat silages, Y28, PA, FA and SB treatments resulted in reductions of total biogenic amines by 55.9, 50.2, 58.9, and 21.1%, respectively, by the end of fermentation compared to the control oat silage. For wilted oat silages, the reduction percentages of total biogenic amine were 40.5, 56.2, 73.0, and 42.7%, respectively, for Y28, PA, FA, and SB treatment by the end of fermentation compared to the control. Clearly, Y28, PA, FA, and SB effectively inhibited biogenic amine formation in oat silage, with the lowest biogenic amine concentrations observed in the FA treatment. Specifically, when FA was applied, only 61.0 mg/kg DM of putrescine and 11.6 mg/kg DM of cadaverine were produced, compared to 173.5 mg/kg DM of putrescine and 350.1 mg/kg DM of cadaverine in the untreated control. Many bacteria genera, such as Bacillus, Clostridium, Escherichia, Proteus, Citrobacter, Klebsiella, Pseudomonas, Salmonella, Shigella, Photobacterium, as well as lactic acid bacteria like Lactobacillus, Pediococcus, Streptococcus, along with certain types of yeast, are known for their ability to decarboxylation amino acid (Pereira et al., 2001; Scherer et al., 2015). FA, PA, and SB are well known for their antimicrobial properties and are commonly used as additives in feed and food preservation (Rooke and Hatfield, 2003). The antimicrobial properties of FA, PA and SB probably inhibited decarboxylase activity, thereby reducing biogenic amines in oat silage.

Biogenic amines and ammonia nitrogen are primarily end products resulting from the degradation of amino acids. Studies by Van Os et al. (1996) and Nishino et al. (2007) have highlighted a strong correlation between ammonia nitrogen content and total biogenic amine concentrations in silages. In this study, a significant correlation (R = 0.49, p < 0.01) was observed between ammonia nitrogen and total biogenic amine concentrations. Ammonia nitrogen is produced through deamination reactions, whereas biogenic amines are generated via decarboxylation reaction (Kung et al., 2003; Xia et al., 2018). In the present study, wilted oat silages exhibited lower levels of total biogenic amines and ammonia nitrogen compared to fresh oat silages. Similar findings of reduced biogenic amine levels in wilted silages have been reported by Steidlová and Kalač (2002). Wilting appears to reduce the concentrations of biogenic amines and ammonia nitrogen in silage, this effect may be attributed to the reduction in water activity of forage. The decrease in water activity may inhibit the activity of both plant-and microbially-derived proteolytic enzymes, which are crucial for the breakdown of proteins into amino acids. This suppression of protein degradation may limit the availability of precursor amino acids for biogenic amine formation during fermentation. Additionally, the lower moisture content may create an environment that is less favorable for the growth and activity of microorganisms responsible for biogenic amine production. High levels of ammonia nitrogen and biogenic amines have been noted in clostridial silages (Tveit et al., 1992). However, the findings from the current study indicate that butyric acid was not detected despite the significant formation of biogenic amines. This suggests that the production of biogenic amines does not necessarily coincide with butyric acid formation.

It has been documented that biogenic amines accumulate in sauerkraut stored at low temperatures (Peñas et al., 2010), and concentrations of cadaverine and putrescine increase in pork meat during low-temperature storage (Halász et al., 1994) However, relatively little attention has been given to biogenic amine formation in silage. Biogenic amine concentrations in silage can indicate undesirable changes in forage (Scherer et al., 2015). In this study, eight biogenic amines (tryptamine, phenethylamine, putrescine, cadaverine, histamine, tyramine, spermidine and spermine) were detected in oat silages fermented at low temperature. Putrescine, cadaverine, and tyramine were the predominant amines, collectively representing approximately 90% of the total biogenic amines measured. Specifically, putrescine, cadaverine and tyramine in oat silages at low temperature were found at concentrations of up to 270.2, 637.9, and 595.7 mg/kg DM, respectively. The levels of biogenic amines observed in the present study exceed those reported by Steidlová and Kalač (2004) for untreated false oat (Arrhenatherum elatius) silages, where putrescine, cadaverine and tyramine levels were 182, 218 and 120 mg/kg DM, respectively. The maximum phenethylamine content in the present study was approximately 23.4 mg/kg DM, similar to levels found in low dry matter perennial ryegrass silage (phenethylamine 17 mg/kg DM) as reported by Van Os et al. (1996). Significant amounts of spermidine (51.5 mg/kg DM) and spermine (24.8 mg/kg DM) were previously detected in untreated oat silage (Křížek, 1993); however, in the present study, only trace amounts of spermidine and spermine were detected in oat silage fermented at low temperature. In the present study, the maximum levels of tryptamine and histamine were approximately 14.1 and 9.2 mg/kg DM, respectively, which are similar to the findings of Scherer et al. (2019), who reported tryptamine and histamine levels of 14.4 and 3.0 mg/kg DM, respectively, in maize var. Tereza silage. Five amines—tryptamine, phenethylamine and histamine, spermidine and spermine—were mostly detected at concentrations below 30 mg/kg DM, with some samples having levels below detection limits. Consequently, the biological effects of these amines on cattle may be considered limited. The type and amount of biogenic amine formation are influenced by various factors, including fermentation temperature, raw material quality, dry matter concentration, the addition of silage additives, and the microorganisms present (Křížek, 1993; Steidlová and Kalač, 2002; Scherer et al., 2019). Currently, there are no regulations governing the biogenic amine content in silages, and there is a lack of information on the minimum concentrations of biogenic amines that may negatively affect animal health. Tveit et al. (1992) found that the dry matter intake (DMI) of early lactation cows decreased when 100 g/day putrescine was infused intraruminally. Scherer et al. (2019) reported no reduction in feed intake in goats within a total biogenic amine concentration range of 1.2 to 4.1 g/kg DM. Van Os et al. (1995) observed that supplementing grass silage was a mixture of biogenic amines at 2.8 g/kg DM tended to reduce the DMI in sheep. Phuntsok et al. (1998) found the ruminal DM digestibility, volatile fatty acids, and intake decreased in steers fed diets that included more alfalfa silage, which contained 2,953 mg/kg DM of putrescine, 3,987 mg/kg DM of cadaverine, 376 mg/kg DM of tryptamine, 2,803 mg/kg DM of phenylethylamine, 12 mg/kg DM of spermine, and 3,078 mg/kg DM of histamine. The maximum total biogenic amine content in our study was 1539.4 mg/kg DM, which is significantly lower than the levels known to reduce voluntary intake of a silage-based diet. However, young animals may be more sensitive to biogenic amines than adults. Fusi et al. (2004) found that supplementing a diet with 1.4 g of mixture biogenic amines per day reduced the DMI and growth rate of weaned Saanen kids. From 30 to 60 days, our study showed an increase in total biogenic amines in both fresh and wilted oat forage. Consequently, the concentrations of total biogenic amines may be higher during later stages of silage feeding than those reported in Tables 7, 8. Therefore, the risk of detrimental health effects on animals consuming oat silages fermented at low temperature cannot be excluded.

In conclusion, the application of Lactobacillus plantarum strain Y28 enhanced the fermentation quality of oat silage at low temperature by decreasing pH and ammonia nitrogen content, while increasing the lactate content. This study also demonstrated that significant amounts of biogenic amines are formed during the low-temperature fermentation of oat silage, with fresh oat silage exhibiting higher levels of biogenic amines than wilted oat silage. The application of Y28, PA, FA and SB effectively inhibited biogenic amine accumulation, thereby improving the safety and quality of oat silage. Among these treatments, FA was the most effective in suppressing the formation of tyramine, cadaverine and putrescine. This research contributes valuable insights into the mechanisms by which L. plantarum Y28 and various additives can modulate the fermentation process and reduce the risk of biogenic amine formation in silage. Furthermore, it opens up new avenues for future research focused on optimizing microbial inoculants and additives to enhance silage preservation, safety, and nutritional quality, particularly in low-temperature environments. Future studies could explore the underlying metabolic pathways involved in biogenic amine production, as well as investigate the long-term effects of such interventions on animal health and productivity.