體外消化與發酵平台應用於豬隻不同飼糧添加物之評估

Abstract

精準餵飼為豬隻生產中降低成本並提高生產效益的重要策略。藉由添加飼糧添加物提高飼糧利用率，並降低豬糞臭味排放是豬隻生產過程相當重要的飼養操作。然而，動物試驗評估飼糧添加物效果成本高且效率低，無法全面分析飼糧在各階段消化的情況。相比之下，體外仿生消化（in vitro bionics digestion）技術具有減少動物使用、高效率、可重複和快速篩選的優勢，且其評估結果與動物體內法（in vivo）高度相關。本實驗室已建立三段式體外消化與發酵平台（three-stage in vitro digestion and fermentation platform），模擬飼糧在胃、小腸消化及大腸發酵的過程，並確立通用的消化條件。接下來，將尋找合適的指標，以全面評估飼糧添加物的效果。 本研究第一部分使用體外消化與發酵平台評估不同飼糧添加物。消化階段以營養分釋放量及消化率為指標，發酵階段以臭味物質、微生物酵素活性、發酵率和產氣動力學為指標。試驗一，利用非澱粉多醣酶（non-starch polysaccharase, NSPase）測試這些評估指標的適用性。結果顯示，藉由分析還原糖、游離胺基酸的釋放量以及營養分消化率，可以評估NSPase添加對提升原料和飼糧營養價值的效果。藉由分析發酵率、產氣動力學和臭味物質，可以評估NSPase添加對飼糧降解程度的影響。消化殘餘物中微生物可利用的基質越少，有助於減少臭味物質的產生。利用不同指標綜合評估飼糧添加物，有助於了解其對豬隻飼糧消化的影響，並提供適當添加濃度及改良建議。然而，NSPase在體外降解飼糧的過程中可能會產生黏性物質，因此應注意飼糧的固液比，以避免營養分釋放效果不佳的問題。試驗二，利用微生物製劑進行體內外試驗。複合型益生菌產品配合母豬餵飼試驗；以粉末或發酵液形式添加的Lactobacillus reuteri分離株R-2和Lactobacillus delbrueckii菌株配合生長豬餵飼試驗，以完成體內外添加效果之雙向評估，驗證體外評估結果之有效性。實驗結果證明，利用體外消化與發酵平台可以釐清飼糧添加物在消化與發酵階段對飼糧的作用機制。在不模擬結腸吸收的情況下，該平台也能評估其對臭味物質產生的潛在影響，並有助於篩選出能夠有效提高飼糧消化率並降低臭味排放的最佳添加濃度。然而，在評估飼糧添加物對採食量、性狀表現、免疫調控、腸道微生物代謝機制、腸道健康等方面的影響時，體外評估仍存在先天的侷限性。飼糧添加物的實際效果仍需要透過動物試驗來驗證。 本研究第二部分延續前一部分試驗二添加不同乳酸菌的餵飼試驗，進一步探討吲哚類化合物的形成途徑。收集豬隻盲腸內容物及糞便，利用LC-MS測定吲哚類化合物的濃度，並結合酵素動力參數進行分析，以釐清微生物代謝色胺酸（tryptophan, Trp）的機制。結果顯示，當Trp含量較低時，可被代謝成吲哚（indole）和吲哚-3-丙酮酸（indole-3-pyruvate, IPyA）；當Trp含量較高時，較可能以Trp →色胺（tryptamine）→ 吲哚-3-乙醛（indole-3-acetaldehyde, IAAld）→ 吲哚-3-乙酸（indole-3-acetate, IAA）→ 糞臭素（skatole）途徑產生糞臭素。因此，盡可能減少消化殘餘物中的Trp含量，可避免Trp被代謝成臭味檢測閾值（odor detection threshold）極低的糞臭素，進一步減少臭味排放。 綜上所述，本研究確定了適合評估飼糧添加物對飼糧消化與臭味排放的指標。體內外雙向評估試驗的結果顯示，進行體外試驗可以探討飼糧添加物對飼糧的作用機制，在一定程度上可以解釋體內試驗反映的結果。希望利用體外消化評估與精簡的動物試驗，完成「預測—偵測—決策」的工作。

Precision nutrition is a crucial strategy in pig production for reducing cost and enhancing production efficiency. Improving feed utilization and reducing pig fecal odor emissions through supplementing feed additives is an important feeding operation in the pig production process. However, evaluating the effects of feed additives using animal experiments is costly and inefficient, and does not allow for a comprehensive analysis of feed digestion at various stages. In contrast, in vitro bionics digestion offers the advantages of reduced animal use, high efficiency, reproducibility, and rapid screening, with the results being highly correlated with the in vivo method. A three-stage in vitro digestion and fermentation platform has been established in our laboratory to simulate the process of feed digestion in the stomach, small intestine and fermentation in the large intestine, and to establish generalized digestion conditions. The next step is to find suitable parameters to fully evaluate the effects of feed additives. In the first part of this study, different feed additives were evaluated using in vitro digestion and fermentation platform. Nutrient release and digestibility were measured in the digestion phase, while odorous compounds, microbial enzyme activity, fermentability and gas production kinetics were measured in the fermentation phase. Trial 1, the applicability of the parameters was tested using non-starch polysaccharidases (NSPase). The results showed that by analyzing the release of reducing sugar, free amino acids, and nutrient digestibility, the effect of NSPase supplementation on enhancing the nutritional value of raw materials and feeds could be evaluated. Additionally, by analyzing fermentability, gas production kinetics and odorous compounds, the impact of NSPase supplementation on feed degradation could be evaluated. The less substrate available to the microorganisms in the digested residue, the fewer odorous compounds are produced. A comprehensive evaluation of feed additives using different parameters can help to understand their impact on pig feed digestion and provide recommendations for appropriate supplemental levels and modifications. However, the in vitro degradation of feed by NSPase may result in the production of sticky substances. Therefore, attention should be paid to the solid-liquid ratio of the feed to avoid poor nutrient release. Trial 2, using microbial addtives in both in vivo and in vitro experiments. A probiotic complex product was supplemented in the sow diet; Lactobacillus reuteri R2 and Lactobacillus delbrueckii strains were supplemented either in powder or fermentation broth in the growing pig diet. Conducting a bidirectional evaluation of the supplemental effects in vitro and in vivo, verifying the reliability of the in vitro results. The experimental results indicated that using the in vitro digestion and fermentation platform could clarify the mechanisms of feed additives during the digestion and fermentation stages. Without simulating colonic absorption, the platform could also evaluate its potential impact on the production of odorous compounds. It assisted in screening out optimal levels of feed additives that can enhance feed digestibility and reduce odor emissions. However, in vitro evaluations have inherent limitations when evaluating the effects of feed additives on feed intake, performance, immune regulation, intestinal microbial metabolism, and gut health. The actual effects of feed additives still require verification through animal experiments. In the second part of this study, it continued the feeding experiments with different Lactobacillus additives from the previous stage to further investigate the pathways of indolic compounds formation. Cecal contents and feces of pigs were collected, and the concentrations of indolic compounds were analyzed using LC-MS, combined with enzyme kinetic parameters to clarify the microbial metabolism mechanism of tryptophan (Trp). The results showed that when the Trp concentration is low, it can be metabolized into indole and indole-3-pyruvate (IPyA); when the Trp concentration is high, it is more likely to follow the pathway of Trp → tryptamine → indole-3-acetaldehyde (IAAld) → indole-3-acetate (IAA) → skatole to produce skatole. Therefore, minimizing the Trp concentration in the digested residues can prevent Trp from being metabolized into skatole, which has a very low odor detection threshold, thereby further reducing odor emissions. In conclusion, this study identified suitable parameters for evaluating the effects of feed additives on feed digestion and odor emissions. The results of bidirectional in vitro and in vivo evaluations indicated that conducting in vitro experiments could elucidate the mechanisms of feed additives on feed, to some extent explaining the results reflected in in vivo experiments. We aim to use streamlined animal experiments alongside in vitro digestion evaluations to complete the "predict-detect-decide" process.