Here again, caryophyllene, piperitone, cis-β-farnesene, and terpinolene found in this study represented the major components of the essential oil of marigold leaves and flowers as well [4,20]. Terpene was the major component of VOCs in marigold flowers which consistent with a previous report and may suggest that high terpene content of VOCs could be the main reason for MCRs’ pungent taste [21].
This study describes the effects of LAB on the biotransformation of VOCs from MCR. These results showed LAB mediated degradation of some terpenes, which agreed with those of a previous study conducted by Figueiredo et al.[22] who found that terpenes in red clover forages decreased greatly after ensilage. Park et al.[19] also found that LAB significantly reduced the terpene content of blueberry juice, including a 92% reduction in vitispirane. The causes of terpene reduction were not fully known, but could involve oxidation to secondary products, glycoside hydrolysis, or ester conversion, as well as isomerization and / or interconversion of some monoterpenols[13,23–25].
However, not all terpene levels were changed. This study has shown that some of the main occurring terpenes were not degraded by LAB, similar to the study of Belviso et al. [26] which showed that while alpha-campholenal can be completely degraded in LAB cultures, alpha-pinene, alpha-terpineol, beta-myrcene, and myrtenal did not degrade at all. Liu et al. [18] reported that some terpenes might be difficult to hydrolyze because their precursors were in the bound form. This could mean that some of these terpenoids present may be in their bound form at the end of the ensilage, or this might be due to enzymatic hydrolysis by glycosidases from microorganisms being limited under the specific conditions found during fermentation (Pogorzelski and Wilkowska, 2007).
An explanation for this late silage degradation of terpenes may be that glycoside precursors were mainly released by acid hydrolysis, a process that occurred slowly[8]. Therefore, terpene levels changing at different times during ensilage could be a result of the different levels of glycoside resistance to acid hydrolysis.
According to the current literature, total terpene content in forage can be reduced by ensilage, but the levels of a small amount of terpenes can increase[22], which is consistent with our results. Similarly, total terpene content declined when LAB was used to ferment berry juice [19], but the total terpene content increased when pomegranate juice was fermented[17]. There is a paucity of information regarding terpene biodegradation by LAB, and studies have shown that terpene biodegradation varies across different species and strains of microorganisms, including LAB. The results from this study have provided preliminary information for future studies on terpene biodegradation in MCR fermentation.
Belviso et al.[26] found that LAB cultures can completely degrade alpha-campholenal and form a new monoterpenoid in 48 hours. Although terpenes are formally composed of one biosynthetic unit, the fact that they can be biotransformed by mechanisms including hydration, isomerization, dehydrogenation, conjugation, oxidation, reduction, decarboxylation, and β-oxidation, means that multiple structures can be produced [2,13]. Microorganisms that promote the biological transformation of terpenes include bacteria, fungi, and yeast. These microorganisms can transform the original terpenes into new ones and other substances via various biotransformation reactions [2,13]. Thus, LAB is responsible for both the degradation of terpenes and the production of the new terpenoidic metabolites. The terpene biotransformation mechanisms of LAB are not well established. Although there have been some reports about the biotransformation activities of LAB during juice and pickle fermentation [16,19], it is difficult to infer the complex relationships between them based on the changes in either the final amount of terpenes or in the kinetics, since there are many other compounds that could interact with terpenes or influence the metabolic behavior of LAB.
Changes in Alcohol levels
These results are consistent with those of Figueiredo et al. [22] who also found that the levels of original alcohols in red clover did not significantly change after ensilage and that some alcohols disappeared.
Wide variations in alcohol levels have been observed for different forage, with comparable or lower concentrations seen in corn, alfalfa, cereal and red clover silages [3,22,27,28]. Current research suggests that a large amount of alcohol is produced during ensilage and that the volatile content of ethanol in corn silage is up to 70% of total VOCs [3,27,28], however, in this study, no ethanol was detected at any stage. Except for ethanol, there is a lack of data on alcohols in silage which are probably generated by amino acid catabolism or by the reduction of aldehydes and ketones [16].
High ethanol contents have been observed in high-dry-matter grass silages due to their high content of fermentable carbohydrates. Low carbohydrate legume forage does not produce more ethanol either [22,28], and silage success is not measured by the production of large quantities of ethanol, which can adversely affect both the environment and the animals themselves [3,27,28].
Acetic acid is the most important organic acid in silage, affects its quality, and is known to possess a sour odor [29]. Acetic acid accumulation depends on substrate supply and the sugar metabolism of the starter culture [29]. In fat metabolism during ensilage, LAB could degrade fatty acids to produce short-chain fatty acids such as butyric acid, acetic acid, butyric acid, and caprylic acid. Goswami et al.[31] found that acetic acid and butyric acid concentrations were significantly increased during the fermentation of horse gram by Lactobacillus plantarum (NRRL-B 4496) and Lactobacillus plantarum (NCDO 1133), indicating that these two strains can effectively metabolize fatty acids to produce short chain fatty acids.
As more acid could be produced in other silage and food fermentation processes, the detection of only two acids in this experiment make this study differ from the rest of the current literature. Since the acids produced by LAB species are strain-dependent [32] , further research is needed on the importance of organic acids to silage quality.
Other VOCs, even at lower concentrations, might considerably influence animal acceptance of forage [33]. In this study, it was not possible to elucidate a clear and definite relationship between MCR ensilage with LAB and VOC biotransformation or to distinguish between the effects of the various VOCs observed. Hence, more research on this specific relationship should be conducted.