優化白肉雞蛋白質體外消化模型

Abstract

蛋白質為白肉雞飼糧中重要的營養分之一，蛋白質的品質取決於胺基酸的平衡以及其消化吸收的效果，有效評估與了解飼料中蛋白質的特性為設計飼料配方前不可或缺的步驟。活體試驗 (In vivo)為了解飼料原料在動物體內消化最真實的方法，然而，執行活體試驗需犧牲動物，也需耗費大量人力、成本與時間。相較之下，體外試驗 (In vitro) 以符合動物體內消化的各項條件，來模擬實際消化道中不同位置的消化，提供一個簡單、快速、重複性高且價格低廉的評估方式。因此，本試驗欲建立白肉雞蛋白質體外消化之模型，作為快速篩選蛋白質原料之平台，並以此平台作為檢測不同飼料原料之消化動力學，藉此瞭解該原料的特性。本研究共分為五個部分。第一部分為挑選快速且便利之體外消化評估方式，以茚三酮(Ninhydrin)與鄰苯二甲醛(o-Phthalaldehyde)反應檢測消化後上清液游離胺基酸之濃度，並以此分析結果與粗蛋白質消化率進行相關性之比較，結果顯示粗蛋白質消化率與 Ninhydrin所測出之游離胺基酸濃度之相關係數為 0.39， 粗蛋白質消化率與 OPA 反應分析之相關係數為 0.34，兩者皆為中度相關，後續試驗會以游離胺基酸釋放濃度與粗蛋白質消化率作為評估之依據。第二部分為建立白肉雞體外消化模型之參數，根據試驗結果並參考其他文獻與考量實驗成本下，釐訂出胃最適合之消化條件為 pH 2.5，每 20 mL 消化液添加胃蛋白酶 67500 units，消化 0.5 小時，而小腸體外消化條件為 pH 7.5，每 40 mL 消化液添加胰酶濃度 0.18 g，消化 3 小時。第三部分為瞭解添加不同動物來源之胰酶對於消化率之影響，在相同胃與小腸消化條件下，添加豬源性商業胰酶所測得之乾物質消化率與粗蛋白質消化率皆顯著高於添加雞來源胰酶之組別，顯示不同動物來源的酵素可能會干擾體外消化效率。第四部分為利用第二部分所建之體外消化模型收集不同消化時長的蛋白質消化率以進行消化動力學之評估，分別評估魚粉、大豆粕，以及由不同比例之玉米、大豆粕與魚粉所配製的飼糧，並了解期消化特性。第五部分，以以糞便或盲腸內容物作為接種液並進行體外發酵，其結果顯示糞便不可取代盲腸內容物作為體外發酵之接種液。而兩者最適合體外發酵之接種物與接種液之比例為 1% S (Substrate, diet) +5% I ( Inocula, cecal contents or feces) 和 1% S+10% I。綜上所述，本研究藉由模擬白肉雞體內的消化道環境，建立一套體外消化模型，提供一個快速、經濟且降低使用實驗動物的替代方式，並釐清不同飼料原料與飼料組成之消化動力學，除此之外，亦了解添加不同接種液對於體外發酵之影響，並釐訂出合適之接種物與接種液之比例。

Protein is one of the most critical nutrients in broiler diets, and its quality is fundamentally determined by amino acid balance and digestive efficiency. Consequently, the effective evaluation and characterization of dietary protein profiles represent an indispensable prerequisite prior to feed formulation design. Although an in vivo experiment is recognized as the gold standard for assessing nutritional quality in poultry by capturing the authentic physiological dynamics within the animal, they inevitably entail animal sacrifice, substantial labor, high costs, and prolonged experimental timeframes. In contrast, in vitro assays simulate digestion across sequential gastrointestinal segments by replicating physiological constraints, thereby offering a simple, rapid, highly reproducible, and cost-effective evaluative platform. Therefore, this study aimed to establish an in vitro protein digestion model for broilers to serve as a rapid-screening platform for protein resources and to characterize the digestive kinetics of diverse feed ingredients to elucidate their distinctive properties.This research was structured into five parts: Part I focused on selecting a rapid and convenient methodology for in vitro digestive assessment. The free amino acid (FAA) concentrations in the post-digestion supernatant were quantified using both ninhydrin and o-phthalaldehyde (OPA) assays, and the analytical outcomes were correlated with crude protein digestibility (CPD). The correlation coefficient between CPD and ninhydrin-detected FAA concentration was 0.39, while that with the OPA reaction was 0.34. Given that both methodologies exhibited moderate correlations, subsequent phases utilized both FAA release concentration and CPD as core evaluative criteria. Part II targeted the parameterization of the in vitro broiler digestion model. Based on the empirical data, literature validation, and economic efficiency, the optimized gastric parameter set was established at pH 2.5 with 67,500 units of pepsin per 20 mL for a 0.5-h incubation, while the optimal small intestinal conditions were designated at pH 7.5 with 0.18 g of pancreatin per 40 mL for a 3-h incubation. Part III investigated the impacts of incorporating pancreatin from porcine and broiler sources. The inclusion of commercial porcine pancreatin yielded significantly higher DMD and CPD compared to the broiler-derived pancreatin group, demonstrating that enzyme origin can critically interfere with in vitro digestive efficiency. Part IV utilized the finalized in vitro model to collect chronological digestibility data for kinetic modeling. Fish meal, soybean meal, and diets formulated with varying blending ratios of corn, soybean meal, and fish meal were evaluated to map the disparities in kinetic profiles and to quantify the respective proportions of rapidly digestible protein (A), slowly digestible protein (B), undigestible protein (UD), and digestible protein (PD) within different matrix compositions. Part V evaluated in vitro hindgut fermentation using either fecal matter or cecal contents as fluid inocula. Fecal matter couldn’t be substituted for cecal contents as an inoculum source to screen and compare gas production across diverse feeds and ingredients. Furthermore, the definitive substrate-to-inoculum ratios were characterized, revealing that ratios of 1% substrate + 5% inoculum and 1% substrate + 10% inoculum were optimally suited for both fecal and cecal fermentation platforms. In conclusion, by simulating the gastrointestinal environment of broilers, this study established a standardized in vitro digestion model, providing a rapid, cost-effective alternative that successfully reduces experimental animal usage. Furthermore, this research elucidated the digestion kinetics of diverse feed ingredients and dietary compositions, clarified the impacts of different inocula on in vitro hindgut fermentation, and determined the optimal operational substrate-to-inoculum ratios.