1、现代食品科技 Modern Food Science and Technology 2023,Vol.39,No.9 23 燕窝唾液酸在大鼠体内的消化、吸收与利用 徐予浛1,谢巧玲1,张薇1,朱梅珍1,陈小旋1,郭东北1,李永忠2,郭建荣2,李红卫1*(1.厦门大学公共卫生学院,福建厦门 361102)(2.小鸟鲜燕锦溢(厦门)健康产业有限公司,福建厦门 361006)摘要:该文研究了含肽即食燕窝、常规炖煮燕窝唾液酸(Sialic Acid,SA)的消化、吸收与利用状况。8 w 龄雄性 SD 大鼠 32只,随机分为空白对照组(Blank Control,BC)、含肽燕窝组(Peptide-Co
2、ntaining Edible Birds Nest,PB)、常规燕窝组(Traditional Edible Birds Nest,TB)、唾液酸标准品组(SA Standard Control,SC),经口灌胃干预。连续2 d 收集粪尿,检测唾液酸吸收量和储留量并计算消化、吸收率;连续 60 d 干预,监测血浆游离、蛋白结合唾液酸、红细胞膜唾液酸含量变化,通过唾液酸化程度评价利用状况。2 d 粪尿监测尚未发现各组唾液酸消化、吸收率有统计学差异。连续 60 d 干预后,各干预组血浆游离唾液酸浓度、血浆蛋白结合唾液酸含量、红细胞膜唾液酸含量(P0.005)均高于 BC 组;干预10 d 时,P
3、B 组蛋白结合唾液酸含量比 BC 组提高 36.36%(P0.05),红细胞膜结合唾液酸比 TB 组、SC 组提高 28.13%和 13.89%(P0.05)。综上,血液的唾液酸化可以反映燕窝唾液酸的利用程度;肽类能促进唾液酸被机体更快速利用;长期食用各类燕窝均能有效提高机体唾液酸化水平且优于摄入游离唾液酸。关键词:N-乙酰神经氨酸;消化;利用;唾液酸化 文章编号:1673-9078(2023)09-23-32 DOI:10.13982/j.mfst.1673-9078.2023.9.1230 Digestion,Absorption,and Utilization of Sialic Aci
4、d from Edible Birds Nests in Rats XU Yuhan1,XIE Qiaoling1,ZHANG Wei1,ZHU Meizhen1,CHEN Xiaoxuan1,GUO Dongbei1,LI Yongzhong2,GUO Jianrong2,LI Hongwei1*(1.School of Public Health,Xiamen University,Xiamen 361102,China)(2.China Fresh Birdie Jinyi(Xiamen)Health Industry Co.Ltd.,Xiamen 361006,China)Abstra
5、ct:The digestion,absorption,and utilization of sialic acid(SA)in the ready-to-eat peptide-containing and the traditional stewed edible birds nest products were compared.Male SD rats aged 8 weeks(n=32)were divided into four groups randomly,including blank control group(BC),peptide-containing edible b
6、irds nest group(PB),traditional edible birds nest group(TB),and sialic acid standard control group(SC).After oral gavage,the feces and urine were collected over 2 days to evaluate the absorption and retention of SA by calculating its digestion and absorption rates.Blood was collected intermittently
7、during 60-day consecutive gavage to monitor the content alteration of the free and protein-bound SA in plasma and the erythrocyte membrane-bound SA,and the degree of sialyation was adopted to evaluate exogenous SA utilization.The results of the 2-day feces and urine monitoring did not reveal any sig
8、nificant statistical differences in SA digestion and absorption among all groups.The results of 60-day consecutive gavage revealed that the free SA concentration(P0.005)and protein-bound SA content(P0.005)in plasma and the erythrocyte membrane-bound SA content(P0.001)were significantly higher in eac
9、h intervention group compared to the BC group.After 10 days of intervention,the protein-bound SA content in the PB group was 36.36%higher than that in the BC group(P0.05),and the erythrocyte membrane-bound SA in the PB group increased by 28.13%and 13.89%compared to the TB and SC groups(P0.05);BC 为空白
10、对照组,PB 为含肽燕窝组,TB 为常规燕窝组,SC 为唾液酸标准品组。现代食品科技 Modern Food Science and Technology 2023,Vol.39,No.9 27 干预开始前(0 wk)大鼠体质量236.02 g,各组无统计学差异(F=1.96,P0.05)。60 d 的干预期间,各组大鼠间的体质量始终保持相近(P0.05)。重复测量方差分析中,体质量不满足球形分布假设(2=531.49,P0.001)。采用 Greenhouse&Geisser方法校正后的结果表明,随干预时间推移,大鼠体质量显著增长,60 d 时整体水平已增长至498.83 g,干预与时间的交
11、互作用无统计学意义(F=0.48,P0.05),各组在 060 d 内体质量增长趋势基本一致。图 2 干预期间大鼠体质量变化 Fig.2 Body weight alteration in rats during intervention 2.2 唾液酸消化、吸收率 图 3 大鼠唾液酸摄入、吸收与储留量 Fig.3 Intake,absorption,and retention of sialic acid 表 3 大鼠唾液酸摄入、吸收与储留量 Table 3 Intake,absorption,and retention of sialic acid (s,n=8,mg/2 d)Group
12、Intake Absorption Retention BC 00-0.390.96-0.852.85 PB 45.970.13 41.967.40 36.1810.98TB 45.180.19 43.721.34 35.925.41SC 45.790.19 43.191.12 36.884.41鉴于绝大多数唾液酸均以原型形态参与肠内吸收或随粪尿排出10,参考氮平衡实验模式,本研究建立“唾液酸平衡”评价不同类型燕窝唾液酸的消化、吸收情况。结果如图 3 所示,在两日的代谢监测期内,由于各组动物体质量、受试物折合唾液酸含量均不同,各组唾液酸摄入量略有差异,BC 组无外源性唾液酸摄入。根据粪尿中唾液
13、酸量对吸收与储留唾液酸量进行推算,计算与分析结果显示,PB 组的唾液酸摄入、吸收、储留量依次为每两日 45.97、41.96、36.18 mg,TB 组依次为每两日 45.18、43.72、35.92 mg,SC 组依次为每两日 45.79、43.19、36.88 mg,不同干预组的唾液酸吸收与储留量虽数值上有所差异,但无统计学意义(P0.05)。(a)各干预物中唾液酸真消化率与表观消化率 (b)各干预物中唾液酸生物价(BV)(c)各干预物中唾液酸净利用率 图 4 不同干预物中唾液酸的消化、吸收率 Fig.4 Digestibility and absorption of sialic aci
14、d in different interventions(n=8)注:图中具有相同上标字母的组间没有显著差异,P0.05。现代食品科技 Modern Food Science and Technology 2023,Vol.39,No.9 28 表 4 唾液酸消化、吸收率 Table 4 Digestion and absorption of sialic acid(s,n=8,wt%)Group Apparent digestibility True digestibilityProtein Biological ValueNet Protein Utilization PB 91.2616
15、.09a 91.2616.09a 86.0319.56a 78.6723.89a TB 97.513.86a 98.283.03a 82.5611.24a 81.3012.24a SC 98.652.56a 98.652.56a 85.5411.44a 84.2310.07a 注:同列右肩相同上标字母表示没有显著差异(P0.05);PB 为含肽燕窝组,TB 为常规燕窝组,SC 为唾液酸标准品组。表 5 各组血浆游离唾液酸浓度变化 Table 5 The change in plasma free sialic acid concentration during the intervention
16、(s,n=8,g/L)Group 0 d 10 d 20 d 30 d 40 d 50 d 60 d BC 0.300.07a10.220.05b 0.360.03b0.320.08b0.310.09b0.330.03c 0.310.08c PB 0.330.08a0.300.04a 0.400.01a0.390.07a0.410.11a0.440.05b 0.550.06ab TB 0.330.06a0.290.03a 0.380.04ab0.390.04a0.370.08ab0.470.02ab 0.450.17b SC 0.290.11a0.310.04a 0.400.03a0.350.
17、05ab0.350.05ab0.480.04a 0.620.08a 注:同列右肩完全不同上标字母表示具有显著差异(P0.05),按均值大小按照 a、b、c、d 以此类推进行标记;BC 为空白对照组,PB 为含肽燕窝组,TB 为常规燕窝组,SC 为唾液酸标准品组。图 4 所示为各干预组唾液酸的表观及真消化率、生物价、净利用率。由于尚未检出大鼠存在内源性的粪代谢唾液酸(TB 组检出少量),故目前实验结果表明唾液酸在机体内的表观消化率与真消化率相近。各干预组表观消化率(F=1.05,P0.05)、生物价(F=1.34,P0.05)、净利用率(F=0.22,P0.05)差异均无统计学意义。故尚未从本研
18、究所构建的“唾液酸平衡”中观察到唾液酸标准品、含肽燕窝、常规燕窝中唾液酸的消化率、生物价或净利用率存在明显不同。推测原因有以下两点:一为该平衡实验为短期干预,短时间内外源唾液酸难以实现体内的驻留;二为该方法精确度低,不适用于评价唾液酸此类含量较低生物活性物质的消化吸收。2.3 血浆游离唾液酸浓度变化 图 5 各组血浆游离唾液酸浓度变化 Fig.5 The concentration of free sialic acid in the plasma in each group 注:*表示该时间点各干预受试物的简单效应具有统计学意义,*表示 P0.05;*表示P0.001。实验进一步通过血液指标
19、考察唾液酸的利用情况。干预期间血浆游离唾液酸浓度的变化如表 5 所示。血液是外源性唾液酸进入人体被吸收、分布和消除的必经之路,红细胞生命周期短且更新代谢快,可更早、更直接反映外源性唾液酸对机体的影响23-25。在长期连续灌胃实验中,除 BC 组以外,各干预组的血浆游离唾液酸、蛋白结合唾液酸与红细胞结合唾液酸含量均显著升高,因此通过监测血液中唾液酸含量变化来评估外源唾液酸的利用程度是可行的。干预前大鼠血浆游离唾液酸浓度整体水平为 0.31 g/L,经 60 d 干预后,干预组浓度比 BC 组提高了约 45.16%100.00%。对大鼠血清游离唾液酸浓度进行重复测量方差分析,数据不符合球形分布假设
20、(2=94.41,P0.001)。Greenhouse-Geisser 的检验结果显示,时间与不同干预物的交互效应显著(F=10.04,P0.001)。简单效应分析结果显示,实验初始(0 d)时,干预受试物的简单效应无统计学意义(F=1.21,P0.05);自给予受试物干预 10 d 开始至 60 d 干预结束,绝大多数时间点的干预受试物的简单效应具有统计学意义(P0.05),在 40 d 时干预物简单效应无统计学意义(P0.05)。多重比较发现,此期间(1060 d)PB 组浓度始终高于 BC 组(P0.05);SC 组在 50 60 d 时间点的血浆游离唾液酸浓度超过两组燕窝干预组(P0.
21、05)。在干预 10 d 时 PB 组、TB 组和 SC 组的血浆游离唾液酸浓度均比初始含量有所提高 (P0.05),并持续升高。时间的简单效应在各干预组都具有统计学意义(P0.05),但并非持续升高,不同干预组均在某些时间点血浆游离唾液酸浓度达到高峰,并开始回落,可能是频繁采血造成的贫血所致。现代食品科技 Modern Food Science and Technology 2023,Vol.39,No.9 29 2.4 血浆蛋白结合唾液酸含量变化 唾液酸的修饰对象包括血浆脂蛋白、血液中多种蛋白因子、细胞表面蛋白和糖脂分子等。干预期间大鼠血浆蛋白结合唾液酸含量如表 6 所示,干预前大鼠血浆蛋
22、白结合唾液酸含量整体水平为 2.14 g/L,经 60 d 干预后,干预组浓度比 BC 组提高了约28.32%56.99%。结合本章 2.3 的结果可见,游离唾液酸浓度上升幅度小,血浆蛋白结合唾液酸则相对稳定上升。游离唾液酸在体内滞留时间短,可能被快速排出体外,或进一步被唾液酸转移酶修饰于大分子表面,从而转为结合形态间接被机体利用。血浆中蛋白结合唾液酸含量是游离唾液酸的 10 倍左右,是极具观测意义的指标。图 6 各组血浆蛋白结合唾液酸含量变化 Fig.6 The content of protein-bound sialic acid in the plasma in each group
23、注:*表示该时间点各干预受试物的简单效应具有统计学意义,*表示P0.05;*表示P0.001。表 6 各组血浆蛋白结合唾液酸含量变化 Table 6 The change in plasma protein-bound sialic acid content during the intervention(s,n=8,g/L)Group 0 d 10 d 20 d 30 d 40 d 50 d 60 d BC 2.160.59a1 2.060.27b 2.410.56a2.420.16b2.920.09c3.020.18c 2.790.33c PB 2.130.59a 2.730.48a 2.
24、820.01a3.080.24a3.860.33a4.310.40a 4.090.29a TB 2.150.47a 2.340.32b 2.330.77a2.560.42b3.370.48b4.150.46ab 3.580.53b SC 2.110.46a 2.550.23ab 2.540.96a2.680.28b3.360.46b3.910.40b 4.380.28a 注:同列右肩完全不同上标字母表示具有显著差异(P0.05),按均值大小按照 a、b、c、d 以此类推进行标记;BC 为空白对照组,PB 为含肽燕窝组,TB 为常规燕窝组,SC 为唾液酸标准品组。对大鼠血浆蛋白结合唾液酸含量进行
25、重复测量方差分析,数据不符合球形分布假设(2=107.88,P0.001)。Greenhouse-Geisser 的检验结果显示,时间与不同干预物的交互效应显著(F=12.29,P0.001)。简单效应分析结果显示,在干预 10 d 以及 30 d 往后的所有时间点,干预物的简单效应均存在统计学意义 (P0.005)。多重比较发现,PB、SC 组大鼠的血浆蛋白结合唾液酸含量较早高于了BC 组,在10 d 时PB组蛋白结合唾液酸含量已较 BC 组提高约 32.52%(P0.05),TB 组在 40 d 时方较 BC 组提高 27.74%;给予干预 10 d 起 PB 组的含量最先高于 TB 组与
26、 SC组(P0.05);在干预后期,各干预组的血浆蛋白结合唾液酸含量逐渐趋于一致,在 60 d 时干预组整体水品达到 4.02 g/L,显著高于此时的 BC 组 2.79 g/L。时间的简单效应在各干预组都具有统计学意义(P0.05),30 d 及往后时间点血浆蛋白结合唾液酸含量均高于 0 d 时的初始含量。目前研究已发现血浆载脂蛋白 A-II、B-48、B-100、C-II、C-III、D、E、J均存在唾液酸化修饰26-28;高密度脂蛋白和低密度脂蛋白的去唾液酸化与动脉粥样硬化、2 型糖尿病的发生有显著相关性29-31,蛋白表面唾液酸的修饰水平影响着机体的健康状况。2.5 红细胞膜结合唾液酸
27、含量变化 图 7 各组红细胞膜结合唾液酸含量变化 Fig.7 The content of erythrocyte membrane-bound sialic acid in each group 注:*表示该时间点各干预受试物的简单效应具有统计学意义,*表示P0.05;*表示P0.001)。干预前大鼠红细胞膜结合唾液酸含量整体水平为0.25 mg/mg prot,经60 d 干预后,干预组浓度比 BC组提高了约 37.21%。唾液酸被认为能够影响红细胞的形态、携氧能力、膜变形性、氧合能力,甚至血红蛋白(Hb)分子的结构和分布32-34,16。有报道称酶法去除红细胞膜表面部分唾液酸后,红细胞聚
28、集程度增加,现代食品科技 Modern Food Science and Technology 2023,Vol.39,No.9 30 且生化特性与细胞存活时间都受到影响。红细胞的抗聚集能力主要源于其自身的电负性,而红细胞 90%的负电荷均为唾液酸所提供35。因此,红细胞表面唾液酸的准确表达对生理和病理过程至关重要36。表 7 干预期间各组红细胞膜结合唾液酸含量变化 Table 7 The change in erythrocyte membrane-bound sialic acid content during the intervention(s,n=8,mg/mg pro)Group
29、0 d 10 d 20 d 30 d 40 d 50 d 60 d BC 0.280.14a1 0.180.05d 0.290.12b0.340.04b0.290.12b0.290.03c 0.430.06b PB 0.250.02a 0.410.03a 0.470.01a0.480.03a0.590.25a0.540.06a 0.60 0.01a TB 0.210.03a 0.320.02c 0.410.07a0.460.10a0.480.08a0.510.02a 0.590.13a SC 0.270.04a 0.360.01b 0.410.08ab0.430.04a0.460.06a0.4
30、30.04b 0.590.08a 注:同列右肩完全不同上标字母表示具有显著差异(P0.05),按均值大小按照 a、b、c、d 以此类推进行标记;BC 为空白对照组,PB 为含肽燕窝组,TB 为常规燕窝组,SC 为唾液酸标准品组。对大鼠红细胞膜结合唾液酸含量进行重复测量方差分析,数据不符合球形分布假设(2=95.34,P0.001)。Greenhouse-Geisser 的检验结果显示,时间与不同干预物的交互效应显著(F=5.30,P0.001)。简单效应分析结果显示,从给予受试物干预 10 d 开始至 60 d 干预结束,受试物的简单效应始终具有统计学意义(P0.005)。多重比较发现,106
31、0 d 各干预组红细胞膜结合唾液酸含量均高于 BC 组(P0.05);干预组中的 PB 组含量均值高于其他组,在 10 d 时与TB 组、SC 组差异具有统计学意义(P0.05);SC 组的红细胞膜结合唾液酸含量在 10 d、50 d 这些时间点上都显著低于 PB 组与 TB 组(P0.05)。PB 组、TB组和SC组的红细胞膜结合唾液酸含量值均在干预10 d时与初始含量出现显著差异(P0.05),并持续升高,干预结束时干预组整体水平达到0.59 mg/mg prot,显著高于 BC 组的 0.43 mg/mg prot。结合 2.4、2.5 的结果,可认为在长期唾液酸负荷下,血液中蛋白与红细
32、胞的唾液酸化程度能够显著提高。时间效应,即受试物长期干预所提供唾液酸的累积,是最主要的效应。5060 d 血液唾液酸含量下降,可能是频繁采血致贫血的影响,而 4050 d 期间血液唾液酸指标基本达到高峰,建议该方法评价实验以4050 d 为宜。蛋白质被吸收前,需在体内消化酶的作用下依次分解为多肽、寡肽、游离氨基酸。除游离氨基酸外,寡肽也被证实能在肠道直接吸收37,并参与机体组织蛋白的再生与合成38,39。寡肽被认为能够起载体作用,将食物中的营养物质运载输送到机体各细胞、组织与器官,促进其他营养成分的吸收。本次受试燕窝的蛋白质消化、吸收率未观察到明显差异,故文中不予赘述。但长期干预过程中,含肽的
33、 PB 组血浆蛋白结合唾液酸、红细胞膜结合唾液酸含量较早出现升高现象,在多个时间点均高于 TB、SC 组。因此推测燕窝肽可能有利于唾液酸分子穿过肠道粘膜细胞,或促进燕窝中唾液酸的利用,在机体内更早实现高水平的唾液酸化。在干预 60 d 时 TB 组的血浆蛋白结合唾液酸、红细胞膜唾液酸含量已与 PB 组接近,说明在保证长期摄入的前提下,各类燕窝产品均能起到提高机体唾液酸化程度的作用,无论是否加入燕窝肽类物质。且在一定时刻,机体唾液酸化水平会达到某种饱和状态,推测机体本身可能存在唾液酸的平衡机制,唾液酸化修饰存在饱和状态。因此外源性唾液酸的补充应遵循长期且适量的原则。3 结论 燕窝的摄入提高了机体
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