CN115590111A - 海洋益生元半乳岩藻聚糖的制备方法及应用 - Google Patents
海洋益生元半乳岩藻聚糖的制备方法及应用 Download PDFInfo
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Abstract
本发明公开了一种海洋益生元半乳岩藻聚糖的制备方法及应用。半乳岩藻聚糖的制备方法，①乙醇脱色；②酶解法提取多糖：纤维素酶、果胶酶、胰蛋白酶的质量比例为2:2:1，维持pH 5.71、温度52℃提取35min，离心浓缩粗多糖液，透析，烘干后得到鼠尾藻粗多糖；③分离并纯化鼠尾藻粗多糖。一种犬猫肠道保健食品，配方包括以下质量份数的原料，鸡腿肉70‑90份、半乳岩藻聚糖0.2‑0.8份、低聚果糖0.5‑1份、甘露寡糖0.1‑0.5份、酿酒酵母提取物1‑3份、丁酸钠0.01‑0.05份、溶菌酶0.5‑1份、维生素B族和胆碱复合物0.1‑0.5份、维生素E 0.1‑0.5份；其中半乳岩藻聚糖不超过总质量的0.8%，低聚果糖、甘露寡糖之和不超过口腔护理配方总质量的1%。本发明能够有效促进肠道益生菌定植，提高猫肠道免疫能力。
Description
技术领域
本发明属于犬猫肠道保健食品技术领域，特别是涉及一种海洋益生元半乳岩藻聚糖的提纯技术及其在调节犬猫肠道菌群平衡和肠道免疫上的应用。
背景技术
动物的肠道菌群研究已经持续了几十年，在小鼠、鸡鸭、伴侣动物、猪牛羊、人等各种动物上均有一定的研究成果。不同动物的肠道菌群组成结构差异巨大，影响肠道菌群差异的原因除了物种本身之外，还受食物的影响，例如外源益生菌、益生元、多糖、短链脂肪酸（SCFAs）等都会影响肠道菌群的结构以及丰富度，除了食物之外，药物、年龄变化、环境变化以及情绪都能通过影响肠道菌群进而影响机体的免疫、代谢等功能。
近年来多糖的研究越来越多，多糖按照来源可分为植物性多糖、动物性多糖以及微生物多糖等，研究较多的是植物性多糖。例如在小鼠模型中，β-(1,3)(1,4)葡聚糖的摄入能够降低小鼠肠道上皮细胞中炎症因子TNF-α以及INF-γ的表达量，此外饲喂富含多糖的食物能改变人类肠道固有菌群的结构。对犬猫来说，目前的研究主要是膳食纤维类型的非结构性多糖，例如黑色亚麻籽多糖（Black psyllium）在肠道内发酵程度较低，导致其利用度较低，主要发挥膳食纤维的生理功能。益生性的结构性多糖则研究较少，只有少量关于果寡糖、甘露寡糖和半乳寡糖等的研究，例如猫每天摄入含0.75%果寡糖的猫粮，能够减少粪便中大肠杆菌的浓度，并且增加双歧杆菌和乳酸菌等益生菌的浓度，表明能够改善猫的肠道菌群。不过犬摄入添加0.5%果寡糖的犬粮后，粪便中乳酸菌以及双歧杆菌的浓度无明显变化，说明犬猫对摄入同样多糖后所受益的菌群略有不同，而且不是所有多糖都有改善肠道功能的作用，有些多糖反而会增加猫的消化负担，猫的胰腺中没有乳糖酶和蔗糖酶，小肠黏膜中的麦芽糖酶的活性很低，所以猫对这些多糖的利用率均较低。另外，不同多糖调控肠道菌群的机制也尚未研究全面，通过调控肠道pH值、抑制有害菌活性增加有益菌数量、增加肠道屏障功能等因素均能改变肠道菌群，因此关于多糖与肠道的关系仍需深入研究，此外海洋益生元多糖对犬猫肠道健康的研究则更少。总体来说，小分子的结构性多糖具有可以被益生菌直接利用以及发挥免疫功能等特点，所以益生性的结构性多糖对犬猫肠道的研究具有市场应用的前景。
犬猫常见的健康问题包括软便腹泻、泌尿肾脏、口腔炎症、肥胖等，针对犬猫常见的健康问题尤其肠道问题开发新型特定的多糖将有助于提升犬猫的生命质量和健康水平。多糖的来源十分广泛，目前宠物市场使用的多糖多为果寡糖、半乳寡糖等，主要来自于植物或者奶制品，而关于海洋益生元例如海藻等来源的多糖研究则较少。
CN113041175A公开了一种源自鼠尾藻的半乳岩藻聚糖及其在犬猫口腔护理上的应用，但是未涉及半乳岩藻聚糖在犬猫肠道的功能作用。
发明内容
为了开发新来源的多糖提取物，本发明的目的是提供一种海洋益生元半乳岩藻聚糖的制备方法及应用，可用于调节肠道菌群平衡和肠道免疫。
一种海洋益生元半乳岩藻聚糖的制备方法，
①乙醇脱色：利用85%乙醇对鼠尾藻进行脱色处理，脱色三次后得到色素较低的乙醇；
②酶解法提取多糖：纤维素酶、果胶酶、胰蛋白酶的质量比例为2:2:1，维持pH5.71、温度52℃提取35min，将含有粗多糖的上清液转移到离心管，利用8000rpm离心7分钟浓缩粗多糖液，并使用3600Da透析袋对上清液透析24小时，烘干后得到鼠尾藻粗多糖；
③分离并纯化鼠尾藻粗多糖：利用DEAE Sephadex A25和 Sephadex G-200 柱，依次使用 0，0.1，0.3 M 的 NaCl 溶液洗脱样品，流速为 0.5mL/min，脱硫、烘干得到纯化后的半乳岩藻聚糖。
一种犬猫肠道保健食品，配方包括以下质量份数的原料，鸡腿肉70-90份、半乳岩藻聚糖0.2-0.8份、低聚果糖0.5-1份、甘露寡糖0.1-0.5份、酿酒酵母提取物1-3份、丁酸钠0.01-0.05份、溶菌酶0.5-1份、维生素B族和胆碱复合物0.1-0.5份、维生素E 0.1-0.5份；其中半乳岩藻聚糖不超过总质量的0.8%，低聚果糖、甘露寡糖之和不超过口腔护理配方总质量的1%；所述的半乳岩藻聚糖根据所述的制备方法得到。
所述维生素B族的组分包括维生素B1、维生素B2、维生素B6和维生素B12 。
一种所述的犬猫肠道保健食品的制备方法，冷冻鸡腿肉于-7~5℃低温解冻12h，将解冻后的鸡腿肉滚揉切块，与半乳岩藻聚糖、低聚果糖、甘露寡糖、酿酒酵母提取物、丁酸钠、溶菌酶、维生素B族和胆碱复合物、矿物质拌匀成型；将混合好的鸡腿肉进行速冻，温度为零下35~零下25℃，时间为0.5-6h，即可得到冻干产品，最后进行紫外杀菌，杀菌时间1min。
本发明的有益效果：
1）优化了半乳岩藻聚糖的制备方法；拓展了其应用；
2）该肠道保健食品配方制成的产品，有助于调节肠道菌群平衡并增强肠道免疫能力，预防犬猫腹泻，减少软便，增强肠道屏障的稳态。具体表现如下：海洋益生元半乳岩藻聚糖作为肠道有益菌的生长原料，通过促进肠道有益菌的定植，来调控菌群平衡，并且增强小肠上皮细胞的抗氧化功能改善肠道免疫能力，来预防腹泻与肠道疾病。半乳岩藻聚糖与低聚果糖、甘露寡糖共同组成复合型益生元，对肠道健康的调节能力更强，酿酒酵母提取物能够改善小肠运动和消化功能，溶菌酶具备杀菌的功能，维生素B族可以增强肠道免疫力。具体如表1：
附图说明
图1是海洋益生元半乳岩藻聚糖的红外光谱检测图。
图2是海洋益生元半乳岩藻聚糖的电喷雾质谱检测图。
图3是海洋益生元半乳岩藻聚糖的电磁共振检测图。
图4是海洋益生元半乳岩藻聚糖（英文名：Fucoidan）的预测结构图。
图5是海洋益生元半乳岩藻聚糖对氧化应激状态下IPEC-J2细胞凋亡过程的影响。
图6海洋益生元半乳岩藻聚糖对氧化应激状态下IPEC-J2细胞丙二醛(MDA)及超氧化物歧化酶(SOD)活性的影响。
图7是扫描电镜（SEM）观察海洋益生元半乳岩藻聚糖对氧化应激状态下IPEC-J2细胞形态的影响。
图8是海洋益生元半乳岩藻聚糖处理 IPEC-J2细胞能够提高铁蛋白(FTH1)和抗氧化蛋白谷胱甘肽过氧化物酶 4(GPX4)表达，并降低铁自噬蛋白(NCOA4、LC3)的蛋白表达量。
图9是海洋益生元半乳岩藻聚糖处理 IPEC-J2细胞能够提高铁蛋白(FTH1)和抗氧化蛋白谷胱甘肽过氧化物酶 4(GPX4) mRNA表达，并降低铁自噬蛋白(NCOA4、LC3)的mRNA表达量。
图10是免疫荧光检测海洋益生元半乳岩藻聚糖处理后 IPEC-J2 细胞内铁自噬蛋白 NCOA4表达下降 (NCOA4:绿色荧光) 。
具体实施方式
以下结合附图和实施例对本发明做进一步的阐述。
实施例1、海洋益生元半乳岩藻聚糖提取以及结构分析
①乙醇脱色：利用85%乙醇对鼠尾藻进行脱色处理，脱色三次后得到色素较低的乙醇。②酶解法提取多糖：纤维素酶、果胶酶、胰蛋白酶比例为2:2:1，维持pH 5.71、温度52℃提取35min，将含有粗多糖的上清液转移到试管中，利用8000rpm离心7分钟浓缩粗多糖液，并使用3600Da透析袋对上清液透析24小时，烘干后得到鼠尾藻粗多糖。③分离并纯化鼠尾藻粗多糖：利用DEAE Sephadex A25（3 × 30 cm）和和 Sephadex G-200 柱，依次使用 0，0.1，0.3 M 的 NaCl 溶液洗脱样品，流速为 0.5mL/min，烘干得到纯化后的半乳岩藻聚糖。经组分分析后发现，主要成分甲基戊糖和D-半乳糖，由此提纯并命名为海洋益生元半乳岩藻聚糖；④脱硫处理：将半乳岩藻聚糖溶于超纯水并使用阳离子交换树脂水解2小时，而后冻干后得到脱硫半乳岩藻聚糖；⑤海洋益生元半乳岩藻聚糖结构分析：用红外光谱、电喷雾质谱检测、核磁共振检测等方法分析半乳岩藻聚糖结构。
试验结果如下，综合红外光谱(图1)、电喷雾质谱(图2)和核磁共振图谱(图3)结果分析，FT-DSG具有典型的半乳糖和岩藻糖骨架以及硫酸化的岩藻低聚糖支链 ，FT-DSG主链为1,3-α-L-岩藻糖和1,6-α-D-半乳糖交替连接构成，主要单元为岩藻糖。图4为FT-DSG的结构预测图。一般方法提取的天然海藻多糖因其独特的理化性质和复杂的组成成分使得其结构较难解析，采用水提法制得鼠尾藻粗多糖，并使用阴离子交换色谱法脱硫处理得到的半乳岩藻聚糖FT-DSG结构更加简单，更加稳定，方便进行结构分析和相应的抗氧化功能研究。
实施例2、不同浓度的海洋益生元半乳岩藻聚糖的肠道保健功能检测
肠道保健食品的制备方法如下：冷冻鸡腿肉于-7~5℃低温解冻12h，将解冻后的鸡腿肉滚揉切块，与半乳岩藻聚糖、低聚果糖、甘露寡糖、酿酒酵母提取物、丁酸钠、溶菌酶、维生素B族和胆碱复合物、维生素E拌匀成型。将混合好的鸡腿肉进行速冻，温度为-35~25℃，时间为0.5-6h，即可得到冻干产品，最后进行紫外杀菌，杀菌时间1min，而后将样品常温避光保存，保持微量元素活性。
样品配方一：鸡腿肉96份、半乳岩藻聚糖0.5份、低聚果糖0.5份、甘露寡糖0.2份、酿酒酵母提取物1份、丁酸钠0.02份、溶菌酶0.5份、维生素B族和胆碱复合物0.3份、维生素E0.3份。
样品配方二：鸡腿肉94份、半乳岩藻聚糖0.8份、低聚果糖0.8份、甘露寡糖0.3份、酿酒酵母提取物2份、丁酸钠0.03-0.05份、溶菌酶0.8份、维生素B族和胆碱复合物0.3份、维生素E 0.3份。
样品配方三：鸡腿肉92份、半乳岩藻聚糖1份、低聚果糖1份、甘露寡糖0.5份、酿酒酵母提取物3份、丁酸钠0.05份、溶菌酶1份、维生素B族和胆碱复合物0.3份、维生素E 0.3份。
选取12只具有频繁软便问题的成年英国短毛去势公猫作为测试对象（体重 4.94± 0.30 公斤），试验环境控制为16小时光照，8小时黑暗的循环节律，每天饲喂10g冻干样品，饲喂周期为21天，7天适应性喂养，14天实验阶段，自由采水。适应性喂养结束，从Day0天（喂食前）起于无菌条件下采集实验猫粪便，并于14天实验期结束后再次收集粪便。在排便后15分钟内采集、形态评分并称重，于-20°C保存。
粪便评分标准：1 = 小颗粒、干硬颗粒；2 = 坚硬、成型的粪便；3 = 柔软、成型且略有潮湿的粪便，形态完整；4 =半固态的不成型粪便；5 =水样液体粪便。
粪便pH测定方法：称取三组定量的粪便，每克粪便稀释于15mL去离子水中混合均匀， 13000 g 离心2 min，利用pH计测定粪便pH值。
粪便干物质测定：称取定量样品，于105℃烘箱中干燥4h，取出于密闭容器冷却30min再行称重并计算。
粪便中氨含量的测定：称取三组100mg粪便分别溶于5mL水中混合均匀，13000 g离心20min后取上清液，配制溶液A（10g/L苯酚和0.05g/L亚硝基铁氰化钠）和溶液B（5g/L氢氧化钠和0.42g/L次氯酸钠）。向每1mL样品中连续加入5mL溶液1和5mL溶液2，于涡旋振荡机上充分混匀，室温反应45min，测定635nm处的吸光值。
粪便中吲哚与3-甲基吲哚含量的测定：利用气象色谱仪对粪便中吲哚含量进行测定，使用三氯甲烷和丙酮提取粪便中的吲哚，采用CBP10-M25-025适应毛细血管柱于180摄氏度检测。
试验效果如下：
1）适口性与采食情况：配方一产品硬度适中，碰撞过程中不会分散，形状保持良好，产品的适口性较好，12只英短均会主动采食冻干；配方二产品硬度较高，结块扎实，形状良好，产品适口性较好，均主动采食；配方三产品硬度较低，容易分散，适口性较低，采食速度较慢，频率较低。
2）粪便形态评分（表2）：
配方一受试猫软便均有不同程度的减轻，粪便形态呈现柔软、成型的条状形态；配方二受试猫的软便情况得到改善，部分受试猫则重新出现软便甚至腹泻的情况；配方三受试猫的粪便形态呈现柔软、成型的条状形态，但部分猫在便后肛门处容易有残留粪便，粪便粘度和湿度较高。
pH值、干物质、氮和吲哚含量（表2）：配方一、二、三受试猫粪便中的pH值、干物质含量（DM）无显著差异；饲喂配方一能显著降低粪便干物质中的氮含量与吲哚含量，粪便的质量较高，成形较好。以上结果表明0.5%添加量的半乳岩藻聚糖软粮产品对猫的消化与排便有一定的改善作用，能够促进肠道消化吸收并改善粪便质量。
表2. 饲喂三种配方的粪便中猫粪便评分、pH值、干物质含量以及氨和吲哚含量
| 配方一 | 配方二 | 配方三 | SEM | P-value | |
| 粪便评分 | 3.0<sup>b</sup> | 2.1<sup>c</sup> | 3.7<sup>a</sup> | 0.1 | 0.037 |
| pH值 | 6.2 | 6.4 | 6.7 | 0.2 | 0.761 |
| DM (%) | 35.1 | 37.5 | 32.1 | 3.6 | 0.627 |
| 氮 (mmol/g DM) | 0.2<sup>B</sup> | 0.4<sup>A</sup> | 0.4<sup>A</sup> | 0.0 | 0.003 |
| 吲哚 (μmol/g DM) | 1.1<sup>C</sup> | 1.7<sup>B</sup> | 2.4<sup>A</sup> | 0.2 | 0.051 |
实施例3、海洋益生元半乳岩藻聚糖调控肠道菌群稳态的功能检测
粪便菌群的分析方法如下：①收集实例2中的粪便样品，于-20℃保存。②粪便样品于55°C的烘箱内干燥后，使用2毫米筛网进行研磨。③总RNA提取。使用DNA 提取试剂盒（QIAamp，Qiagen，Valencia，CA）完成DNA的提取，并利用分光光度计测定 DNA 的数量和质量。④荧光定量PCR引物合成。合成双歧杆菌，乳酸杆菌属、大肠杆菌和产气荚膜梭菌的荧光定量PCR引物。⑤荧光定量PCR测定。使用384孔透明光学反应板，每孔包括5.5 μL 2× SYBRGreen PCR混合染料，0.4 μL 上下游引物，2 μL DNA （2 ng），和 2.7 μL无菌水。使用Applied Biosystems实时荧光定量PCR仪器进行测定。
试验效果如下：①粪便中微生物基因表达：配方一的猫粪中双歧杆菌含量显著增加，乳酸菌、大肠杆菌没有显著差异，产气荚膜梭菌显著降低；配方二的猫粪中双歧杆菌含量显著增加，同时大肠杆菌的含量也有显著提升，肠道菌群较为紊乱；配方三的猫粪中双歧杆菌、乳酸菌、大肠杆菌和产气荚膜梭菌的含量没有显著差异（图6）。以上结果表明添加量的半乳岩藻聚糖冻干产品对猫肠道菌群有一定的调控作用，并能够通过增多肠道益生菌的数量以达到改善软便和增强消化功能的作用。
表3是饲喂海洋益生元半乳岩藻聚糖后猫粪中双歧杆菌，乳酸杆菌属、大肠杆菌和产气荚膜梭菌的含量变化。
表3.饲喂三种配方的粪便中肠道菌群的基因表达量
| 空白组 | 配方一 | 配方二 | 配方三 | SEM | P-value | |
| 双歧杆菌 | 10.2 | 13.5<sup>A</sup> | 12.3<sup>C</sup> | 10.7<sup>A</sup> | 0.4 | <0.001 |
| 乳酸杆菌 | 10.5 | 10.3 | 10.6 | 10.4 | 0.2 | 0.571 |
| 大肠杆菌 | 8.7<sup>B</sup> | 8.6<sup>B</sup> | 9.7<sup>A</sup> | 8.5<sup>B</sup> | 0.2 | <0.001 |
| 产气荚膜梭菌 | 11.2<sup>a</sup> | 7.2<sup>b</sup> | 10.4<sup>a</sup> | 11.4<sup>a</sup> | 0.3 | <0.001 |
实施例4、海洋益生元半乳岩藻聚糖的肠道免疫与抗氧化功能检测
氧化应激细胞模型建立过程：①细胞氧化应激模型建立：选取哺乳动物猪肠道上皮细胞系IPEC-J2（Porcine intestinal epithelial cells）用作抗氧化应激的细胞炎症试验模型，利用梯度浓度的H2O2处和DEM（0-4 mM）一起孵育，使用DMEM/F-12 混合洗涤缓冲液清洗两次，使IPEC-J2产生氧化应激反应，建立细胞水平的氧化应激模型。②样品处理：组分1为无处理的IPEC-J2细胞；组分2为氧化应激模型细胞；组分3为抗氧化剂Trolox处理的氧化应激模型细胞；组分4为半乳岩藻聚糖处理的氧化应激模型细胞。分别收集四个组分的细胞样品。
细胞凋亡检测：将上述四个组份细胞样品加入70%的乙醇4℃过夜固定，配制终浓度为100μg/mL的PI染液，4℃避光染色4组细胞样品，利用流式细胞仪于激光光波波长为488nm，发射光波波长为630nm的参数下进行检测，并对数据进行分析。
①丙二醛（MDA）检测：利用硫代巴比妥酸(thiobarbituric acid，TBA)处理样品，使用分光光度计分别检测并计算532nm与600nm吸光度的差值，计算得出丙二醛的含量。②超氧化物歧化酶（SOD）检测：利用黄嘌呤及黄嘌呤氧化酶反应系统37℃水浴加热样品，560nm处测定各样品吸光值，计算得出超氧化物歧化酶的含量。
铁蛋白(FTH1)、抗氧化蛋白谷胱甘肽过氧化物酶 4(GPX4)和铁自噬蛋白(NCOA4、LC3)的蛋白含量检测：使用细胞裂解液裂解细胞，1200rpm离心10min，缓冲液与样品按照4:1的比例混匀，100℃变性10min，制成蛋白质样品。Western-blot检测样品的蛋白质含量。
免疫荧光检测铁自噬蛋白 NCOA4的表达情况：利用 4％ 的甲醛对预处理的样品4℃过夜固定，利用NCOA4抗体进行免疫染色并拍照。
试验结果如下，① 细胞凋亡检测结果：抗氧化剂Trolox和半乳岩藻聚糖能显著缓解氧化应激细胞的细胞凋亡效果（图5）。②丙二醛（MDA）检测结果：抗氧化剂Trolox和半乳岩藻聚糖能降低氧化应激细胞的丙二醛表达，显著降低细胞的过氧化水平（图6）。③超氧化物歧化酶（SOD）检测结果：抗氧化剂Trolox和半乳岩藻聚糖均能降低氧化应激细胞的超氧化物歧化酶的含量，显著缓解氧化应激情况（图6）。④机制研究：为探究半乳岩藻聚糖的抗氧化机理，首先使用扫描电镜观察半乳岩藻聚糖处理后的细胞形态（注：IPEC-J2本身细胞为长条梭型，经扫描电镜前处理后细胞变成圆球形），发现半乳岩藻聚糖能够显著细胞的病变，即皱缩情况明显改善（图7）；为探究线粒体形态的回复是否通过抑制铁自噬通路实现，利用Western-blot检测细胞的铁自噬相关蛋白水平，研究发现半乳岩藻聚糖与Trolox能够显著提高铁蛋白(FTH1)和抗氧化蛋白谷胱甘肽过氧化物酶 4(GPX4)表达，并降低铁自噬蛋白(NCOA4、LC3)的蛋白表达量与mRNA表达量（图8、图9）；通过免疫荧光实验再次验证了半乳岩藻聚糖与Trolox对氧化应激细胞内铁自噬蛋白 NCOA4的抑制作用（图10）。以上实验结果表明，半乳岩藻聚糖具有良好的抗氧化应激与消炎功能，其作用机制是通过抑制肠道上皮氧化应激细胞内的铁自噬发生，缓解线粒体形态，以恢复氧化应激造成的细胞损伤，能够有效地发挥肠道保健功能。
上述试验结果和实例是对本发明的具体描述，但不局限于以上的具体实施方式。本发明中的试验结果仅仅是部分配方的展示和产品形态的表现，实际操作过程不限制于这些配比和形态，可能以多种形式展现，例如冻干、粉剂、胶囊、液体等。此外还可能在上述基础上进行更多的形态变化和改动，本领域的普通技术人员能从本发明公开的内容直接导出或联想到的所有变形，均应认为是本发明的保护范围。本发明的保护范围由所附权利要求及其任何等同物给出。
Claims (4)
1.一种海洋益生元半乳岩藻聚糖的制备方法，其特征在于：
①乙醇脱色：利用85%乙醇对鼠尾藻进行脱色处理，脱色三次后得到色素较低的乙醇；
②酶解法提取多糖：纤维素酶、果胶酶、胰蛋白酶的质量比例为2:2:1，维持pH 5.71、温度52℃提取35min，将含有粗多糖的上清液转移到离心管，利用8000rpm离心7分钟浓缩粗多糖液，并使用3600Da透析袋对上清液透析24小时，烘干后得到鼠尾藻粗多糖；
③分离并纯化鼠尾藻粗多糖：利用DEAE Sephadex A25和 Sephadex G-200 柱，依次使用 0，0.1，0.3 M 的 NaCl 溶液洗脱样品，流速为 0.5mL/min，脱硫、烘干得到纯化后的半乳岩藻聚糖。
2.一种犬猫肠道保健食品，其特征在于：配方包括以下质量份数的原料，鸡腿肉70-90份、半乳岩藻聚糖0.2-0.8份、低聚果糖0.5-1份、甘露寡糖0.1-0.5份、酿酒酵母提取物1-3份、丁酸钠0.01-0.05份、溶菌酶0.5-1份、维生素B族和胆碱复合物0.1-0.5份、维生素E0.1-0.5份；其中半乳岩藻聚糖不超过总质量的0.8%，低聚果糖、甘露寡糖之和不超过口腔护理配方总质量的1%；所述的半乳岩藻聚糖根据权利要求1所述的制备方法得到。
3.根据权利要求2所述的犬猫肠道保健食品，其特征在于：所述维生素B族的组分包括维生素B1、维生素B2、维生素B6和维生素B12 。
4.一种根据权利要求书2所述的犬猫肠道保健食品的制备方法，其特征在于：冷冻鸡腿肉于-7~5℃低温解冻12h，将解冻后的鸡腿肉滚揉切块，与半乳岩藻聚糖、低聚果糖、甘露寡糖、酿酒酵母提取物、丁酸钠、溶菌酶、维生素B族和胆碱复合物、矿物质拌匀成型；将混合好的鸡腿肉进行速冻，温度为零下35~零下25℃，时间为0.5-6h，即可得到冻干产品，最后进行紫外杀菌，杀菌时间1min。
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| CN114747688A (zh) * | 2022-04-19 | 2022-07-15 | 深圳市豆柴宠物用品有限公司 | 一种具有改善犬猫肠道功能的“四位一体”的组合添加剂及其制备方法 |
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| CN113754788A (zh) * | 2021-09-28 | 2021-12-07 | 上海海洋大学 | 一种海蒿子岩藻聚糖及其制备方法和应用 |
| CN114747688A (zh) * | 2022-04-19 | 2022-07-15 | 深圳市豆柴宠物用品有限公司 | 一种具有改善犬猫肠道功能的“四位一体”的组合添加剂及其制备方法 |
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| CN114276958A (zh) * | 2021-12-20 | 2022-04-05 | 无锡弘焕微生态科技有限公司 | 具有抗炎功效的三重益生菌发酵复合物的制备方法和应用 |
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