Evaluation of the potential of non-starch soluble polysaccharides from 3 algae to modulate the immune response and metabolic state in the intestine

Non-starch soluble polysaccharides (NSPs) produced by yeasts are used in animal nutrition to improve health and performance. However, the magnitude of the effect may be dependent upon the quantity and the composition of the polysaccharides. As seaweeds are attractive sources of NSPs, this study was set up to evaluate their potential to improve intestinal health. The effect of NSP extracts prepared from Saccharomyces cerevisiae containing β-glucan and mannan (PSY1, positive control) or a mixture of mannanoligosaccharides (PSY2, positive control), micro algae containing β-glucan (PSA1), brown macro algae containing fucoidan and laminarin (PSA2), and green macro algae containing ulvan (PSA3) on intestinal porcine epithelial cells J2 (IPEC-J2) was studied in the presence and absence of the enterotoxigenic bacterium Escherichia coli k99 strain (ETEC) as an in vitro challenge. The E.coli-k99 strain with adhesion factor F41 (41/32) was isolated from a mastitis-infected udder. In addition, a mixed extract prepared from vegatal orgin supplemented with phenolic compounds from vegetal origin, zinc and selenium (9631), and ZnO were tested to compare responses to NSP extracts. Gene expression was measured in IPEC-J2 cells after 2 and 6 hours of incubation using “whole genome” porcine microarrays (submission as a conference paper at the SEAGRICULTURE 2017 6th International Seaweed Conference). IPEC-J2 cells were grown in 2 cm2 wells for 7 days at 37 ºC and 5% CO2 using 1:1 DMEM/Ham’s F10 1:1 medium supplemented with 5% FCS without antibiotics. For all tests, confluent monolayers were washed twice with medium without FCS (hereafter denoted as medium) and incubated for 1 hour with this medium. Hereafter, the medium was discarded and an extract dissolved/suspended in medium was added. Different concentrations of extracts and ZnO (concentrations are specified in the source name column) were used for incubation of IPEC-J2 monolayers for a period of 2 and 6 hours in the absence and presence of ETEC. All incubations were tested in duplicate and for each type of additive duplicate control wells containing no additive (only culture medium) were incubated for 2 and 6 hours. After incubation total RNA was extracted. RNA's extracted from replicates were pooled (biological replicates) and this pool was hybridized in duplicate (technical replicates). IPEC-J2 enterocyte cell line derived from the jejunum of piglets, host-feed interaction

酵母源非淀粉可溶性多糖（Non-starch soluble polysaccharides, NSPs）常用于动物营养领域，以改善动物健康与生产性能。然而，其作用效果的强弱可能取决于多糖的添加量与组成结构。鉴于海藻是NSPs的优质来源，本研究旨在评估其改善肠道健康的潜力。
本研究针对猪小肠上皮细胞J2（intestinal porcine epithelial cells J2, IPEC-J2），探究了多种NSP提取物的作用效果，包括含β-葡聚糖（β-glucan）与甘露聚糖（mannan）的酿酒酵母（Saccharomyces cerevisiae）提取物（PSY1，阳性对照）、低聚甘露糖（mannanoligosaccharides）混合物（PSY2，阳性对照）、含β-葡聚糖的微藻（micro algae）提取物（PSA1）、含岩藻聚糖（fucoidan）与昆布多糖（laminarin）的褐藻（brown macro algae）提取物（PSA2）以及含藻聚糖（ulvan）的绿藻（green macro algae）提取物（PSA3）；实验设置了产肠毒素大肠杆菌K99菌株（enterotoxigenic bacterium Escherichia coli k99 strain, ETEC）体外攻毒与非攻毒两种处理条件。
本研究使用的大肠杆菌K99菌株带有黏附因子F41（41/32），分离自患乳腺炎的乳腺组织。此外，本研究还测试了植物源混合提取物（添加有植物源酚类物质、锌与硒，编号9631）以及氧化锌（ZnO），以对比其与NSP提取物的细胞响应差异。
于孵育2小时和6小时后，采用猪全基因组微阵列（whole genome porcine microarrays）对IPEC-J2细胞的基因表达水平进行检测（相关内容已作为会议论文提交至2017年第六届国际海藻会议SEAGRICULTURE 2017）。IPEC-J2细胞于2 cm²培养孔中培养7天，培养条件为37℃、5% CO₂，培养基采用1:1混合的达尔伯克改良伊格尔培养基（Dulbecco's Modified Eagle Medium, DMEM）与Ham’s F10培养基，并添加5%胎牛血清（fetal calf serum, FCS）且不含抗生素。
所有实验均采用汇合单层细胞：先用无FCS的培养基（后续简称基础培养基）洗涤两次，再用该培养基孵育1小时。随后弃去培养基，加入溶解/悬浮于基础培养基中的提取物。本研究设置了不同浓度的提取物与ZnO（浓度详见样品名称列），用于IPEC-J2汇合单层细胞的孵育，孵育时长分为2小时与6小时，同时设置攻毒与非攻毒两组处理。
所有孵育实验均设置重复孔；每种添加剂均设置不含添加剂的空白对照孔（仅含基础培养基），同样孵育2小时与6小时，且均为重复孔。孵育结束后提取总RNA。将重复孔提取的RNA混合（生物学重复），随后将混合后的RNA进行重复杂交（技术重复）。
IPEC-J2细胞系为仔猪空肠来源的肠上皮细胞系，用于宿主-饲料互作研究。