改善破壁製程以提升酵母菌細胞壁水解產物之腸黏膜免疫功效

2020 年全球酵母市場價值估計為 39 億美元,預計到 2025 年將達到 61 億美元。其中,細胞壁是生產酵母提取物的副產品,含有 55-60% β-1,3-1,6-葡聚醣和30-40%甘露醣蛋白,已被證實具有刺激先天性免疫系統的潛在能力,又稱為生物活性調節劑(biological response modifier),能直接與先天性免疫細胞進行交互作用, 並活化促進下游相關免疫反應。通過適當控制裂解條件之破壁製程,不僅可以獲得分離後的酵母提取物和細胞壁碎片,還可以修飾細胞壁以增強其免疫刺激功能。本研究的目的是建立一種破壁加工製程,以生產可有效刺激免疫反應的酵母細胞壁產物。本研究使用自體水解(胞內酵素)和酵素水解(商業外添酵素)來實現細胞壁裂解製程。第一部分探討自體水解製程(Autolysis),使用醋酸鹽調節 pH 至 4.5, 釋放的氨基酸量高於用鹽酸調節 pH 值的傳統自解製程,胞內酵素水解能力能使細胞壁造成孔洞及塌陷。為了增進破壁效果,並修飾細胞壁結構,再利用添加內切 β-葡聚醣酶 Viscozyme 並結合自體水解進行破壁製程(Combined Autolysis-Hydrolyis process, CAH)。結果發現,在自解製程進行後 6 小時及 12 小時再介入酵素(CAH 6h 及 CAH 12h)相較於一開始添加(CAH 0h)可以使胞內核苷酸釋出量增加,並且使平均細胞壁碎片粒徑減少。這可能是由於自解會先使細胞壁機械強度減弱,使外添要素更易進行水解。然而,使酵素更容易作用反而可能造成更多細胞壁 β-葡聚醣的損失,使其 β-葡聚醣含量在四組別中最低。此結果也反應在模擬腸道免疫試驗中,CAH 12h 組的 IL-6 釋放量最低;而含有最多 β-葡聚醣含量的 CAH 0h 組則顯著高於其他組別,顯示 β-葡聚醣含量為一個免疫刺激重要的評估指標,也顯示酵母細胞壁多醣的各種物化性質對於刺激免疫效果會有不同的影響,包括溶解度、粒子大小、β-glucan 含量等。然而,所有樣品均對刺激 Raw264.7 分泌細胞因子有顯著影響,且釋放量都顯著低於 LPS 組,因此本研究之細胞壁破壁產品都具有作為安全且溫和之腸道黏膜免疫刺激膳食補充劑之潛力。

關鍵字

酵母細胞壁； β-葡聚醣；腸道免疫；自體水解；酵素水解； Raw 264.7 ；細胞激素

並列摘要

The global yeast market is estimated to be valued at USD 3.9 billion in 2020 and is projected to reach USD 6.1 billion by 2025. Among the yeast product, high-valued yeast extract plays a vital part in food additive and nutritional supplements. However, the cell wall, which is the by-product of yeast extract production containing 55-60% β-1,3-1,6-glucan and 30-40% mannoprotein, is proved to be a biological response modifier (BRM) that could stimulate innate immune system. Via cell wall lysis process, we can obtain yeast extract and cell wall after separation. By proper control of the lysis conditions, the cell wall could also be modified to enhance its immunostimulatory function. The purpose of this study was to establish a lysis procedure to produce a yeast cell wall product that could effectively stimulate immune reaction. In this study we used conventional lysis process, autolysis (endogenous enzyme), and enzyme hydrolysis (exogenous enzyme) to achieve cell wall lysis. The experimental results showed that by using acetate to adjust pH to 4.5, the amount of released amino acid was higher than that of traditional autolysis process, which used hydrochloric acid for pH adjustment. We found that the endogenous enzyme could make cell wall porous and subside. In order to increase the degree of lysis and to modify cell wall structure as well, we combined viscozyme (endo-beta glucanase) and autolysis process to lysis cell wall. It was found that adding enzyme after 6 and 12 hours autolysis processing (CAH 6h CAH 12h) increased the amount of RNA released and decreased particle size as compared with addition exogeneous enzyme at the beginning (CAH 0h) of process. Autolysis may undermine cell wall strength, making glucanase effectively break down the cell wall but meanwhile lowering the content of β-glucan. We further compared the immunostimulatory function of autolysed and combined hydrolysed cell wall products by in vitro intestinal mucosa immunity test. The result indicated that the CAH 12h samples which contain minimum β-glucan content showed the lowest secretion of cytokine by Raw264.7; while the CAH 0h, the highest β-glucan content, showed the highest secretion, confirming that β-glucan content may be the main factor for stimulating immune reaction. Results of this study also indicated that various physicochemical properties of the yeast cell wall polysaccharides have different effects on immunostimulation, including solubility, particle size, β-glucan content, etc. However, all samples prepared in this study had significant effect on stimulating Raw264.7 to secrete cytokines, and the amounts of cytokines released were significantly lower than that of the LPS group. Therefore, the cell wall lysis products prepared in this study may have potential to be safe and useful dietary supplements for benefiting intestinal mucosa immune system.

並列關鍵字

yeast cell wall ； yeast β-glucan ； mucosa immunity ； autolysis ； enzyme hydrolysis ； Raw 264.7 ； cytokines

參考文獻

Aguilar‐Uscanga, B., and J. M. Francois. A study of the yeast cell wall composition and structure in response to growth conditions and mode of cultivation. 2003 Letters