Grass carp (Ctenopharyngodon idellus) is one of the most crucial freshwater aquaculture species in China, and crisp grass carp (C. idellus C.et V) is its high-value commodity product. In the early 1970s, fish farmers accidentally discovered that muscle textural quality (hardness, springiness, etc.) of grass carp was improved significantly after being fed solely with faba bean (Vicia faba, FB) for 90–120 days, and this variety was called crisp grass carp (Yu et al., 2017). The muscle textual changes of crisp grass carp were found to associate with the structure of muscle fibers and collagen contents rather than the proximate composition(Wang et al., 2015; Ma et al., 2020a). Because of the high market value,crisp grass carp has been protected as the “China Geographical Indication Product”, and its products are exported to North America (Yang et al., 2015). However, a large amount of faba bean intake causes poor palatability, intestinal inflammation, oxidative damage and growth retardation of grass carp (Li et al., 2018; Zhang et al., 2015). To alleviate these negative impacts of faba bean, two previous studies tested the effect of FB extracts on grass carp, demonstrating that FB water extract improved the textural quality with little or no negative effects on grass carp physiology (Ma et al., 2020a, 2020b). FB water and alcohol extracts also promoted growth performance, indicating the possibility that functionally active components of faba bean can be isolated by a simple extraction procedure. However, these studies had a limitation that the formulated diets were not isonitrogenous or isoenergetic. This limitation causes the uncertainty about effectiveness and the safety evaluation of FB extracts as aquafeed additives.

The intestinal microbiota plays a crucial role in many important physiological processes of the fish host including growth, metabolism and immunity (Fetissov, 2017). The abundance of beneficial and harmful bacteria in the intestine is closely related to the health of the host (Hornef, 2015). Microbiota regulates intestinal absorption and extraintestinal metabolism of fatty acids in the zebrafish (Semova et al.,2012). In grass carp, intestinal microbiota is known to help digest polysaccharose, contributing to nutritional and physiological homeostasis(Ni et al., 2013). In a study on leopard coral grouper (Plectropomus leopardus), Firmicutes and Fusobacteria had been shown to influence the hydrolysis of maltose and trehalose (Mekuchi et al., 2018). Moreover, it was reported in grass carp that Bacillus subtilis modulated the bacterial assembly, lipid metabolism and immune response in the intestine (Shi et al., 2020). These findings led to the application of probiotics, in which the modulation of microbiota improved immunity and growth in rainbow trout (Oncorhynchus mykiss) (Adel et al., 2017). On the contrary, disruption of intestinal microbial community weakens immunity and increases the risk of disease in fish (Tran et al., 2018). So, assessment of intestinal microbiota is essential for the evaluation of effectivenessand safety of faba bean and their extracts. In order to achieve comprehensive evaluation of effectiveness and safety of FB extracts as aquafeed additives, this study used four types of FB extracts including FB water extract (FBW), FB alcohol extract (FBA), FB protein (FBP), and FB residues (FBR) prepared according to our previous report (Ma et al., 2020a). The active components of these four faba bean extracts were firstly analyzed, and then these extracts were used to formulate isonitrogenous and isoenergetic diets for a feeding trial to examine their effects on growth performance, muscle quality, intestinal microbiota, metabolism, immunity and oxidative responses in grass carp.