克氏原螯虾（Procambarus clarkii）作为我国重要的淡水养殖虾类之一，有着良好的养殖前景。鱼粉作为水产饲料中的组成部分，以其良好的适口性和丰富均衡的营养元素等优势，成为水产饲料中重要的蛋白源之一。然而受到国际形势和环境要素等影响，鱼粉的价格不断上涨，导致克氏原螯虾的养殖利润和产业发展受到不利影响。因此，开发我国资源丰富、价格低廉的动植物蛋白源部分或完全替代鱼粉是打破制约我国水产养殖业发展瓶颈的重要举措。然而，动植物蛋白源部分或完全替代鱼粉会影响水产养殖动物肌肉的品质与风味，这方面与克氏原螯虾相关的调控研究还十分有限，涉及到作用机制的研究也鲜见报道。因此，本试验旨在探究全动植物蛋白源对克氏原螯虾肌肉品质和风味的影响，进一步基于“肠-肌”轴明确肠道微生物及其代谢产物对其肌肉品质影响的机制，同时评估茶树精油和丁酸梭菌对其风味的调控功能和机制，为提升克氏原螯虾肌肉品质的功能饲料配方的设计和改良提供研究思路、理论依据和发展方向。本试验包含以下几个部分：

1 全动植物蛋白源对克氏原螯虾生长和肌肉品质的影响

本试验旨在评估不同全植物蛋白源与全动物蛋白源对克氏原螯虾生长性能、肌纤维发育、肌肉抗氧化和肌肉蛋白质沉积的影响。本试验分别以豆粕、菜粕等全植物饲料原料与肉骨粉、血粉等全动物饲料原料作为两组饲料的蛋白源，设计并使用了两组等氮等能的试验饲料，分别命名为PP组与AP组，进行了为期8周的养殖试验。结果表明，全植物蛋白源喂养的克氏原螯虾（PP组）饵料系数显著降低，肌肉粗蛋白含量显著增加（P < 0.05）。在肌肉质构方面，PP组的克氏原螯虾肌肉剪切力和硬度显著高于AP组，而黏附性显著低于AP组（P < 0.05）。PP组克氏原螯虾肌肉的肌纤维平均直径显著高于AP组。在相关基因表达方面，PP组克氏原螯虾肌肉中蛋白质合成调节因子、肌球蛋白和肌肉生成调节因子较AP组显著上调，MSTN、肌肉蛋白质水解-自噬和泛素化相关因子显著下调（P < 0.05）。在肌肉氧化指标方面，PP组肌肉的蛋白羰基含量显著低于AP组（P < 0.05）。因此，与全动物蛋白源相比，全植物蛋白源对克氏原螯虾的生长、肌肉发育、和肌肉品质具促进作用，这种作用是可能是通过影响MSTN/AKT介导的蛋白质合成和蛋白质水解通路的动态平衡实现的。

2 全动植物蛋白源基于“肠道微生物-TCDCA-MSTN”轴影响克氏原螯虾肌肉蛋白沉积的机制

在试验一的基础上，本试验旨在阐明全动植物蛋白源对克氏原螯虾肌肉生长和蛋白质沉积的影响机制。本试验使用16S rDNA测序和代谢组学分析了克氏原螯虾肠道中微生物及其代谢产物的组成，并使用分子生物学分析了与肌肉生长、蛋白沉积和水解相关基因的表达变化。结果表明，在肠道微生物及其代谢产物方面，PP组Bacteroides丰度显著下调，Anaerorhabdus furcosa丰度显著上调；牛磺鹅脱氧胆酸（Taurochenodeoxycholic acid，TCDCA）等18种代谢产物含量显著升高（P < 0.05），邻氨基苯甲酸和色氨酸酰酪氨酸含量显著下调。差异肠道微生物、差异食糜代谢物和肌肉发育相关差异基因进行相关性分析，结果显示，Bacteroides与TCDCA、维生素B1和维生素D3呈显著负相关，与色氨酸酰酪氨酸和邻氨基苯甲酸呈显著正相关，同时TCDCA与蛋白质合成调节因子和肌肉生成调节因子相关基因的转录丰度呈显著正相关。通过靶向代谢组学对体外厌氧发酵后的克氏原螯虾肠道内容物进行测序分析，结果证明在检测出的胆汁酸种类中TCDCA为含量最高的胆汁酸，且在PP组中显著性上升。因此，与动物蛋白源相比，植物蛋白源对克氏原螯虾肌肉蛋白沉积和肌纤维发育起到了促进的作用，该作用有可能是通过“拟杆菌属Bacteroides-肠道代谢物TCDCA-MSTN介导的蛋白质生成相关基因”这一途径完成的。

进一步在常规饲料中添加300 mg/kg TCDCA，旨在验证TCDCA影响克氏原螯虾肌肉发育和蛋白质代谢的功能。试验结果表明，在全植物蛋白源中添加TCDCA显著提高了克氏原螯虾的末重、增重率和特定生长率以及肌肉中粗蛋白含量（P < 0.05）；肌肉质构方面显著提高了肌肉的剪切力并降低了粘着性；在肌肉组织形态上，TCDCA组肌肉纤维密度和长度显著增加并且与蛋白质合成，及肌纤维发育相关基因的表达水平上调（P < 0.05）。此外，通过KEGG对克氏原螯虾的肠道微生物功能进行分析，结果表明，与代谢途径（蛋白质代谢、脂肪酸代谢、代谢物的生物合成、辅因子和维生素的代谢、外源性生物降解和代谢以及核酸代谢）和消化系统相关的微生物的丰度增加。因此，在饲料中添加TCDCA能够促进克氏原螯虾的生长和肌肉发育，提高与肌肉脂肪酸和蛋白质代谢相关的肠道微生物丰度。

3 基于GC-IMS和人工感官设备分析全动植物蛋白源对克氏原螯虾肌肉风味的影响

除了蛋白质等营养物质的沉积，肌肉的风味与口味也是影响经济价值的重要指标。本试验进一步采用电子舌和气相色谱-离子迁移光谱仪（GC-IMS）技术研究了不同蛋白源组克氏原螯虾蒸煮后肌肉的嗅觉和味觉风味。本实验饲料配方同实验一，分别命名为全植物蛋白源组（PP组）和全动物蛋白源组（AP组），对比了两组克氏原螯虾蒸煮后肌肉的挥发性化合物成分和风味成分。GC-IMS共鉴定出34种挥发性成分，包括醛类、酮类、醇类和酯类。其中3-辛醇（单体）、3-呋喃甲醇（二聚体）、2-甲基-1-戊醇（单体）、2-丙醇（单体）、庚醛（单体）和烯丙酸（单体）在AP组中含量显著高于PP组（P < 0.05），是摄食动物性蛋白源小龙虾肌肉中的标志性挥发性成分。在口感方面，PCA结果证明了两组间口感具有显著性差异（P < 0.05）。电子舌检测出了肌肉的苦味、丰富度、甜味、涩味的回味方面的参数，其中AP组的小龙虾比PP的小龙虾表现出更高的丰富度，这可能与动物蛋白源组肌肉中IMP含量的显著增加有关（P < 0.05）。因此，全动物蛋白源组比全植物蛋白源组的虾味道和气味更加丰富，说明选用植物蛋白源对克氏原螯虾在味道和风味方面产生了负面影响。

4 茶树精油和丁酸梭菌对克氏原螯虾肌肉风味的调控研究

针对试验三研究发现的全植物蛋白源对克氏原螯虾肌肉风味和口感产生了负面影响，本试验计划采用茶树精油和丁酸梭菌作为功能性添加剂缓解全植物蛋白导致的克氏原螯虾风味下降的影响。试验设计五个试验组：全植物蛋白组（PP），全动物蛋白组（AP），在全植物蛋白饲料基础上补充100 mg/kg茶树精油的全植物蛋白（TTO）组，补充50 mg/kg（1×108 CFU/g）丁酸梭菌（CLO）组以及同时补充茶树精油和丁酸梭菌组（TC）。结果表明，与PP组相比，AP组显著降低了克氏原螯虾肌肉CMP水平，增加了AMP、GMP和IMP水平（P < 0.05）；显著增加了肌肉中赖氨酸含量，显著降低谷氨酸、甘氨酸、酪氨酸、蛋氨酸、异亮氨酸、亮氨酸和脯氨酸的含量（P < 0.05）；显著增加C20:1n9、C20:5n3和C22:6n3的含量，显著降低C18:2和C18:3的水平（P < 0.05）；增加了肌肉中的挥发性化合物整体丰度，其中增加了丁酮、戊酮和苯乙酮水平，降低了己酮含量。在PP组中补充TTO显著提高了克氏原螯虾WGR和SGR，降低了FCR（P < 0.05）；增加了肌肉中Asp和His的含量，降低了Ala的含量（P< 0.05）；提高了肌肉鲜味。在PP组基础上添加CLO后Asn和His水平显著升高，而Glu和Met水平显著降低（P < 0.05）；肌肉中C20:1n9的含量TC组Glu含量显著增加（P < 0.05）。与PP组相比，TC组显著增加C17:1和C20:1n8的水平和肌肉鲜味（P < 0.05）。此外，克氏原螯虾肌肉主要的差异风味成分是W5S—氮氧化合物，W1S—甲烷化合物和W2S—醛酮类和醇类。相关性分析结果表明，氨基酸主要与回味B和长链烷烃类呈负相关，但与氮氧化合物呈正相关。核苷酸与苦味、回味B和长链烷烃类等呈负相关，而与丰富度、氮氧化合物和甲烷化合物呈正相关。脂肪酸与甜味和甲烷化合物呈负相关。挥发性化合物主要与甜味、苦味和芳香度呈正相关。因此，在全植物蛋白源饲料中添加茶树精油和丁酸梭菌，对克氏原螯虾的肌肉风味和口感产生了改良作用，这种改良主要是通过改变肌肉中脂肪酸、氨基酸的含量产生的。

Procambarus clarkii as as one of the important freshwater aquaculture shrimps in China, has a good commercial development prospect and broad research value. Fishmeal, as a component of aquafeed, has become one of the important protein sources in aquafeed with the advantages of good palatability and rich and balanced nutritional elements. However, the imbalance between the supply and demand of fishmeal has been aggravated in recent years due to the influence of the international situation and environmental factors, etc. The price of fishmeal has been increasing, which ultimately leads to the profitability of crayfish farming and the development of the industry to be affected by the ineffectiveness of crayfish. Therefore, the development of resourceful and inexpensive animal and plant protein sources to partially or completely replace fishmeal is an important measure to break the bottleneck that restricts the development of China's aquaculture industry. However, the current understanding of the effects of different animal and plant protein sources on muscle quality and flavor of aquatic animals is very limited, and studies involving the mechanism of action have rarely been reported. Therefore, the aim of this study was to investigate the effects of different protein sources on muscle quality and flavor of crayfish, and explore the mechanisms of gut microorganisms and their metabolites on muscle quality based on the gut-muscle axis. Meanwhile, we further assessed the functions and mechanisms of tea tree essential oil and Clostridium butyricum in regulating their flavor. This will provide research ideas, theoretical basis and development direction for the design and improvement of quality-enhancing functional feed formulations for crayfish. This study contains the following parts:

1 Effects of different protein sources on the growth and muscle quality of Procambarus clarkii

The aim of this experiment was to evaluate the effects of different protein sources on growth performance, muscle fiber development, muscle antioxidant properties and muscle protein deposition in crayfish. In this experiment, two groups of experimental feeds with equal nitrogen and energy were designed and used as protein sources for two groups of feeds, respectively, using plant-based feed ingredients such as soybean meal and rapeseed meal, and animal-based feed ingredients such as meat and bone meal and blood meal, which were named as PP group and AP group, respectively, and were cultured for a period of 8 weeks. The results showed that the bait coefficient of crayfish (PP group) fed with plant-based protein sources was significantly lower and muscle crude protein content was significantly increased (P < 0.05). In terms of muscle texture, muscle shear and hardness of crayfish muscle in PP group were significantly higher than that of AP group, while adhesion was significantly lower than that of AP group (P < 0.05). The mean diameter of myofibers of crayfish muscle in PP group was significantly higher than that of AP group. In addition, in terms of related gene expression, protein synthesis regulators, myosin and muscle production regulators were significantly up-regulated and MSTN, muscle protein hydrolysis-autophagy and ubiquitination-related factors were significantly down-regulated in the muscles of crayfish in the PP group compared with the AP group (P < 0.05). In terms of muscle oxidative indices, the protein carbonyl content of muscles in the PP group were significantly lower than those in the AP group (P < 0.05). In summary, compared with the animal protein source, the plant protein source had a certain degree of promoting effect on the growth, muscle development, and muscle quality of crayfish, which was achieved mainly by affecting the dynamic balance of the MSTN/AKT-mediated protein synthesis and protein hydrolysis pathways.

2 Mechanisms of different protein sources affecting muscle protein deposition in Procambarus clarkii based on the “Gut microbes-TCDCA-MSTN” axis 

Based on experiment 1, this experiment aimed to elucidate the mechanism of the effect of different protein sources on muscle growth and protein deposition in crayfish. In this experiment, the composition of microorganisms and their metabolites in the gut of crayfish was analyzed using 16S rDNA sequencing and metabolomics, and the changes in the expression of genes related to muscle growth, protein deposition and hydrolysis were analyzed using molecular biology. The results showed that in terms of gut microorganisms and their metabolites, the abundance of Bacteroides was significantly down-regulated in the PP group and the abundance of Anaerorhabdus furcosa was significantly up-regulated; the content of 18 metabolites, including taurochenodeoxycholic acid (TCDCA), was significantly elevated, and the content of o-amino benzoic acid and tryptophan-tyrosine contents were significantly down-regulated. Correlation analysis of differential gut microorganisms, differential chow metabolites and differential genes related to muscle development showed that Bacteroides was significantly negatively correlated with TCDCA, cholecalciferol and thiamine, and positively correlated with Trp-Tyr and o-aminobenzoic acid, while TCDCA was significantly and positively correlated with the abundance of transcripts of genes related to regulators of protein synthesis and regulators of muscle production. TCDCA was also significantly positively correlated with the transcript abundance of genes related to protein synthesis regulators and muscle production regulators. Sequencing analysis of the intestinal contents of crayfish after in vitro anaerobic fermentation by targeted metabolomics demonstrated that TCDCA was the most abundant bile acid among the bile acid species detected and was significantly increased in the PP group. In conclusion, the experimental results indicate that compared with animal protein sources, plant protein sources promote muscle protein deposition and myofiber development in crayfish, and this effect may be accomplished through the pathway of “Bacteroides-intestinal metabolite TCDCA-MSTN-mediated proteogenesis-related genes” pathway.

Further, by adding 300 mg/kg TCDCA to the regular diet, we aimed to verify that TCDCA can act as a link in the gut-muscle axis to affect the muscle development and protein metabolism of crayfish. The experimental results showed that the addition of TCDCA to plant-based protein sources significantly increased the final weight, weight gain rate and specific growth rate of crayfish in terms of growth, and increased the crude protein content of muscle; increased the shear force and reduced the adhesion of muscle texture; in terms of muscle histomorphology, the muscle fiber density and length of the TCDCA group significantly increased and up-regulated the expression of genes related to protein synthesis and the development of muscle fibers. expression of genes related to protein synthesis, and muscle fiber development. In addition, KEGG analysis of gut microbial function in crayfish showed that the abundance of microorganisms associated with metabolic pathways (protein metabolism, fatty acid metabolism, metabolite biosynthesis, cofactor and vitamin metabolism, exogenous biodegradation and metabolism, and nucleic acid metabolism) and the digestive system was increased. In summary the addition of TCDCA increased growth performance and muscle development and improved the abundance of gut microorganisms associated with muscle fatty acid and protein metabolism in crayfish.

3 GC-IMS and artificial sensory equipment based analysis of the effect of different protein sources on the muscle flavor of crayfish

In addition to the deposition of nutrients such as proteins, the flavor and taste of crayfish muscles are also important indicators of economic value. In this experiment, the olfactory and gustatory flavors of cooked muscles of crayfish fed different protein sources were investigated using electronic tongue and gas chromatography-ion mobility spectrometry (GC-IMS) techniques. Two experimental diets were set up, namely, plant protein source (PP group) and animal protein source group (AP group), and the volatile and flavor components of boiled muscles of crayfish fed the two experimental diets separately were investigated. An artificial sensory device based on sensing technology, e-tongue and e-nose, was used to analyze both flavor and taste of cooked crayfish muscle, and gas chromatography-ion mobility spectrometry (GC-IMS) was used to qualify and quantify the volatile constituents of cooked crayfish muscle. In terms of flavor, the electronic tongue results showed that there were significant differences between the two groups of crayfish steamed muscles in terms of several benzenes, nitrogen oxides, hydrides, methane compounds, aromatic components and organosulfides and long-chain alkanes. A total of 34 volatile components including aldehydes, ketones, alcohols and esters were also identified by GC-IMS. Among them, 3-octanol (monomer), 3-furanmethanol (dimer), 2-methyl-1-pentanol (monomer), 2-propanol (monomer), heptanal (monomer), and allylpropionic acid (monomer), which were significantly higher in the AP than in the PP group, were the hallmark volatile constituents in the muscles of crayfish of animal protein origin for consumption. In terms of taste, the parameters of bitter, richness, sweetness and astringent aftertaste aspects of the muscle were detected by the electronic tongue, in which the crayfish in the AP group showed higher richness than those in the PP group, which may be related to the increase in the IMP content in the muscle of the animal protein source group. The PCA results proved that there was a significant difference in the taste between the two groups. The results of this experiment showed that crayfish that consumed animal protein source contained richer taste and odor than those in the group that consumed -plant protein source, su