Bioefficiency of microencapsulated hemp leaf phytonutrient-based extracts to enhance in vitro rumen fermentation and mitigate methane production

PLOS ONE RESEARCH ARTICLE Bioefficiency of microencapsulated hemp leaf phytonutrient-based extracts to enhance in vitro rumen fermentation and mitigate methane production Srisan Phupaboon ID1, Maharach Matra1, Ronnachai Prommachart1,2, Pajaree Totakul1,3, Metha Wanapat ID1* a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 1 Tropical Feed Resources Research and Development Center (TROFREC), Department of Animal Science, Faculty of Agriculture, Khon Kaen University, Khon Kaen, Thailand, 2 Department of Animal Science, Faculty of Agriculture and Natural Resources, Rajamangala University of Technology, Tawan-Ok, Chonburi, Thailand, 3 Division of Animal Science, Faculty of Agricultural Technology, Rajamangala University of Technology Thanyaburi, Thanyaburi, Pathum Thani, Thailand * OPEN ACCESS Citation: Phupaboon S, Matra M, Prommachart R, Totakul P, Wanapat M (2024) Bioefficiency of microencapsulated hemp leaf phytonutrient-based extracts to enhance in vitro rumen fermentation and mitigate methane production. PLoS ONE 19(10): e0312575. https://doi.org/10.1371/journal. pone.0312575 Editor: Adham A. Al-Sagheer, Zagazig University Faculty of Agriculture, EGYPT Received: October 13, 2023 Accepted: October 10, 2024 Published: October 31, 2024 Copyright: © 2024 Phupaboon et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the manuscript and its Supporting Information files. Funding: The research was financially supported by Fundamental Fund (FF) (no. 65A103000130), Ministry of Higher Education, Science, Research, and Innovation (MHESI), and Khon Kaen University, Thailand. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Abstract The objective was to assess the supplementation with microencapsulation of hemp leaf extract (mHLE) utilized as a rumen enhancer on in vitro rumen fermentation and to enhance the bioavailability of active compounds for antimicrobial action, particularly in protozoa and methanogen populations. The feed treatments were totally randomized in the experimental design, with different levels of mHLE diet supplemented at 0, 4, 6 and 8% of total DM substrate and added to an R:C ratio of 60:40. During fermentation, gas kinetics production, nutrient degradability, ammonia nitrogen concentration, volatile fatty acid (VFA) profiles, methane production, and the microbial population were measured. The supplemented treatment at 6% of total DM substrate affected reductions in gas kinetics, cumulative gas production, and volatile fatty acid profiles, especially the acetate and acetate to propionate ratio. Whereas propionate proportion and total volatile fatty acid concentration were enhanced depending on the increase of nutrients in vitro dry matter degradability (IVDMD) after 12 h of post-fermentation at a R:C ratio of 60:40 (P < 0.05). Consequently, mHLE addition resulted in optimal ruminal pH and increased nutrient degradability, followed by ammonia nitrogen concentrations (P < 0.05), which were enhanced by dominant cellulolytic bacteria, particularly Ruminococcus albus and Ruminococcus flavefaciens, which showed the highest growth rates in the rumen ecology. Therefore, mHLE, a rich phytonutrient feed additive, affected the methanogen population, reduced the calculated methane production and can be a potential supplement in the ruminant diet. Introduction At present, there is interest in an extensive selection of agro-nutrient plants as potential feed additives for reducing the chain of food production’s effects on the environment. Hemp PLOS ONE | https://doi.org/10.1371/journal.pone.0312575 October 31, 2024 1 / 15 PLOS ONE Competing interests: The authors have declared that no competing interests exist. Phupaboon S et al. (Cannabis sativa L.) derivatives are among the agro-nutrient products used in animal nutrition to reduce feed costs and increase the sustainability and quality of animal products, particularly meat and milk [1]. There are suggestions that hemp plant-derived products from agricultural operations can improve meat and/or milk production, shelf life, and animal health [1]. One such example is the rapidly growing hemp sector, which generates seeds, leaves, seed oil, and cake. These hemp by-products can be a valuable source of protein in the diets of ruminants, potentially replacing protein source such as soybean meal [1]. According to several studies on the nutritional value, the crude protein (CP) content obtained from hemp by-products exceeds the recommended dietary needs for ruminant growth (120–180 g/kg CP DM) and maintenance (60–110 g/kg CP DM) [2, 3]. In addition, they provide an essential amino acid profile that is well-balanced and equivalent to soybean meal, particularly in regard to methionine (1.8 and 2.0% CP) and lysine (6.4 and 6.8% CP) as per the body requirements for goats and cattle, respectively [4–6]. Semwogerere et al. [6] have discussed the bioavailability and bioefficacy of hemp phytochemicals for enhancing ruminant health, production, and increasing meat shelf life. Bioavailability of dominant bioactive compounds in hemp by-products, such as terpenoids, alkaloids, flavonoids, terpenes, phenolics, lignans, plant steroids, curcumins, saponins, glucosides, cannabinoids, and polyphenols, is added to the diet of ruminant animals to improve rumen manipulation and decrease methane production during fermentation [7], while tetrahydrocannabinol (THC) and cannabidiol (CBD) are unpublished modes of action in feed additives to mitigate methane. The most abundant secondary phytochemical constituents are phenolics, which account for 45% of all secondary phytochemical constituents in plants, followed by terpenoids and steroids (27%), alkaloids (18%), and others (10%). They have anti-inflammatory, antispasmodic, anti-allergic, antioxidants, antibacterial, antifungal, chemo preventive, neuroprotective, hypotensive, and antiaging properties [6]. The nutritional quality of spent hemp biomass and its implications for animal health, emphasizing the need for further investigation into its cannabinoid content. Industrial hemp is defined as containing CBD (>1%) and THC (0.03%, and while it can yield significant amounts of CBD, the residual cannabinoids, including CBD and THC, may remain in byproducts post-extraction. Specifically, the research indicates that spent hemp biomass can contain residual cannabinoids, which may accumulate in animal tissues and potentially affect consumer exposure [8]. Recent evidence suggests that the in vivo experiment based on the bioactivity profile of their byproducts showed how these plant products can be used (as an additive � 3% of the total DM diet) as supplements in terms of p (...truncated)