Chinese Journal of Animal Nutrition >

2024 , Vol. 36 >Issue 6: 4036 - 4049

DOI: https://doi.org/10.12418/CJAN2024.346

RESEARCH PAPER

Effects of Different Extrusion Puffing Processes on Comprehensive Utilization of Soybean Meal Protein in Vitro

  • ZHANG Siyu , 1 ,
  • ZHANG Shuo 1 ,
  • ZHANG Hongliang 1 ,
  • LI Peiqi 1 ,
  • WANG Xianli 1 ,
  • WU Min 2 ,
  • SUN Chengguo 3 ,
  • HE Yang 1 ,
  • SU Huawei , 1, *
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  • 1 State Key Laboratory of Animal Nutrition and Feeding, College of Animal Science and Technology, China Agricultural University, Beijing 100193, China
  • 2 College of Engineering, China Agricultural University, Beijing 100083, China
  • 3 COFCO Oils R&D Center, Beijing 102209, China
*associate professor, E-mail:

Received date: 2023-12-28

  Online published: 2024-06-07

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Abstract

This study was conducted to investigate the effects of different extrusion puffing processes on the rumen fermentation and small intestinal digestion of soybean meal through a three-step in vitro method. A single-factor randomized trial design was used to prepare the nine samples, which were soybean meal (CON group), extrusion puffing soybean meal (E group), soybean meal adding 3% soybean crude oil before extrusion puffing (AO group), soybean meal adding 3% soybean crude oil after extrusion puffing (CO group), soybean meal adding 3% soybean crude oil at the same time before and after extrusion puffing (AO-CO group), soybean meal adding 4% glucose before extrusion puffing (GE group), soybean meal adding 4% glucose and 3% soybean crude oil before extrusion puffing (GAO group), soybean meal adding 4% glucose before extrusion puffing and 3% soybean crude oil after extrusion puffing (GCO group), and soybean meal adding 4% glucose before extrusion puffing and 3% soybean crude oil at the same time before and after extrusion puffing (GAO-CO group). The properties of the extrudate, such as color difference, degree of browning, and fiber and protein composition, were determined, and the effects of extrusion puffing processes on the degree of Maillard reaction of soybean meal protein were investigated. Then 3 Angus steers with permanent rumen fistulas weighing about 600 kg were selected to estimate the rumen degradation characteristics of soybean meal after extrusion puffing and the small intestinal digestibility of its rumen undegradable protein (RUP) was determined by a three-step in vitro method, which was metabolized by rumen fluid, then pepsin and finally trypsin. The results showed as follows: 1) the results of color difference after extrusion puffing showed that the values of redness (a*), yellowness (b*), color difference (ΔE) and absorbance (OD) at 420 nm in experimental groups were significantly increased compared with CON group (P<0.05), and the brightness (L*) value (except for AO group) was significantly decreased (P<0.05). The OD value at 294 nm in GAO group was significantly lower than that in AO and GE groups which were with only one reactant added (P<0.05), and the values of a* and ΔE were significantly lower than those in GE group (P<0.05). Except for GCO group, the contents of neutral detergent fiber (NDF) and neutral detergent insoluble crude protein (NDICP) in GE group were significantly increased compared with CON group and the other experimental groups (P<0.05). 2) Compared with CON group, the rumen non-degradable rates, small intestinal digestibility in vitro, total digestive tract digestibility in vitro of dry matter (DM) and crude protein (CP) and contents of RUP and small intestine digestible crude protein (IDCP) in experimental groups were significantly increased (P<0.05). The rumen non-degradable rates of DM and CP and contents of RUP and IDCP (except for E group) in GE group were significantly higher than those in the other experimental groups (P<0.05). 3) After 16 h in vitro fermentation, the pH and ammonia nitrogen (NH3-N) content in rumen fluid in experimental groups were significantly lower than those in CON group (P<0.05). Extrusion puffing did not have a significant effect on the total volatile fatty acid content (P>0.05), but changed the volatile fatty acid composition in rumen fluid, in which the contents of isobutyric acid, isovaleric acid and total branched-chain volatile fatty acid in rumen fluid in experimental groups were significantly decreased compared with CON group (P<0.05). In conclusion, the addition of glucose can improve the degree of Maillard reaction during extrusion puffing and promote the digestion and absorption of high-quality protein from soybean meal through rumen to small intestine, but the addition of soybean oil rich in unsaturated fatty acids can inhibit the Maillard reaction during extrusion puffing. Therefore, it is recommended to add glucose during extrusion puffing, while not recommended to add oils rich in unsaturated fatty acids.

Key words： twin-screw extrusion puffing; extrusion puffing soybean meal; Maillard reaction; glucose; soybean crude oil

Cite this article

ZHANG Siyu , ZHANG Shuo , ZHANG Hongliang , LI Peiqi , WANG Xianli , WU Min , SUN Chengguo , HE Yang , SU Huawei . Effects of Different Extrusion Puffing Processes on Comprehensive Utilization of Soybean Meal Protein in Vitro[J]. Chinese Journal of Animal Nutrition, 2024 , 36(6) : 4036 -4049 . DOI: 10.12418/CJAN2024.346

蛋白质饲料在反刍动物体内消化、吸收和利用不同于单胃动物。反刍动物可以从饲粮、微生物蛋白(microprotein,MCP)和内源性氮等不同途径中获取可用于代谢的氮源[1]。饲粮提供的蛋白质可分为瘤胃可降解蛋白质(rumen degradable protein,RDP)和瘤胃不可降解蛋白质(rumen undegradable protein,RUP)。RDP被瘤胃微生物降解后,产生小肽、氨基酸和氨,进一步被微生物合成MCP或者部分通过瘤胃进入小肠[2]。到达小肠的蛋白质来源还包括RUP和内源分泌物以及脱落细胞结合的蛋白质[3]。食糜经过真胃到达小肠后,在消化酶的作用下会被进一步分解为氨基酸,通过肝脏到达肾脏,或者流入血液,未消化的蛋白质则以尿液、粪便的形式排出体外[4]。因此,对于氨基酸组成不平衡的蛋白质饲料,如棉籽粕、花生粕等,微生物降解蛋白质的过程能够提高其蛋白质的利用率。然而,对于富含限制性氨基酸赖氨酸和蛋氨酸,氨基酸平衡的优质蛋白质饲料(如豆粕),微生物降解蛋白质的过程会对其利用效率造成一定程度的浪费,使得流入小肠内的蛋白质不能满足高产反刍动物营养需要。为保障我国粮食安全,达到“提效节粮”的目标,在提高豆粕优质蛋白质的过瘤胃效率的同时,保证蛋白质的小肠利用率已经成为研究人员和畜牧产业亟待解决的问题。
目前,常用于提高蛋白质过瘤胃效率的方法主要有热处理、化学处理以及物理包被等[5-6]。许多研究根据蛋白质高温易变性的特点进一步探索发现[6⇓⇓-9],加热条件下蛋白质发生不可逆的美拉德反应能够降低反刍动物瘤胃中蛋白质的消化率,从而提高豆粕优质蛋白质的过瘤胃效率。美拉德反应是氨基化合物(如蛋白质、肽、氨基酸等)和羰基化合物(如还原糖、脂质、醛、酮等)之间的一类复杂化学反应[10]。王潍波[9]研究证明,葡萄糖添加量、加热温度、加热时间、含水量4个因素均对蛋白质消化率影响显著,豆粕干物质(DM)消化率与瘤胃蛋白质消化率会随着加热温度升高、加热时间延长、葡萄糖添加量增加而逐渐下降。张恺容[11]研究发现,较低水分处理的挤出样品美拉德反应程度更大,瘤胃中蛋白质消化率更低,并且可以通过色差来检测挤出样品的美拉德反应程度。在生产实践中,研究发现在荷斯坦母犊牛开食料中添加挤压膨化豆粕能够提高干物质采食量(dry matter intake,DMI),促进骨骼生长和体重增加[12]。在不影响乳成分和饲料效率的情况下,挤压膨化豆粕能够提高奶牛DMI,促进产奶量增加[13]。Risyahadi等[14]通过Meta分析发现,饲料膨化处理能有效提高奶牛的产奶量和乳糖含量以及DM和蛋白质消化率,但对乳脂和蛋白质含量无显著影响。利用美拉德反应虽然能提高豆粕蛋白质的过瘤胃率,但因加热过久、温度过高可能导致蛋白质在小肠内消化率降低,使一些氨基酸如半胱氨酸、酪氨酸和赖氨酸等受到破坏。因此,需要控制加热条件,减少过度热处理产生的负面影响。
油脂因其具有“高能量”、“易吸收”特点,是解决肉牛快速育肥过程中能量需要简便有效的方法[15]。大豆毛油是大豆经过浸出或压榨后得到的未进行精炼的原油,富含不饱和脂肪酸。虽然过量油脂添加对瘤胃微生物有较强的抑制作用[16],但是研究发现油脂对饲料颗粒具有包被作用,可有效降低优质蛋白质在瘤胃的消化率,提高RUP的小肠消化率[17]。另外,曾泽等[18]研究也表明,体外添加3%大豆油能够调控瘤胃发酵,降低甲烷排放。
因此,本研究以豆粕为原料,加入葡萄糖或大豆毛油,混合均匀后利用双螺杆挤压膨化机进行挤压,冷却后部分样品采用大豆毛油进行包被,测定在不同生产条件下挤出物的美拉德反应程度、瘤胃降解情况以及肠道降解情况,以期能在实际生产中控制反应条件,更好地控制美拉德反应程度,从而提高豆粕优质蛋白质的过瘤胃效率。

1 材料与方法

1.1 试验材料

试验用豆粕和大豆毛油由中粮油脂(菏泽)有限公司提供,其中大豆毛油长链脂肪酸组成见表1;葡萄糖为市售产品,规格为分析纯,纯度≥99.9%。
表1 大豆毛油长链脂肪酸组成

Table 1 Long-chain fatty acid composition in soybean crude oil%

项目
Items		 含量
Content
C12∶0 0.01
C13∶0 <0.01
C14∶0 0.07
C14∶1n5 0.01
C15∶0 0.01
C15∶1n5 0.02
C16∶0 10.51
C16∶1n7c 0.10
C17∶0 0.10
C17∶1n7 0.05
C18∶0 3.74
C18∶1n9t 21.91
C18∶1n9c 0.86
C18∶2n6t 54.17
C18∶2n6c 0.31
C20∶0 0.03
C18∶3n6 0.19
C20∶1 7.10
C18∶3n3 0.04
C21∶0 0.02
C20∶2n6c 0.08
C22∶0 0.31
C20∶3n6 0.01
C22∶1n9 0.01
C20∶3n3 0.01
C23∶0 0.05
C20∶4n6 0.02
C22∶2n6 0.13
C24∶0 0.02
C20∶5n3 0.03
C24∶1n9 0.07
C22∶6n3 0.01
饱和脂肪酸 SFA 14.86
不饱和脂肪酸 UFA 85.14

1.2 试验设计

1.2.1 膨化工艺

膨化前豆粕经过如下条件预处理:粉碎后过2 mm分析筛,添加水或者葡萄糖溶液调节物料含水量至20%,混合均匀。
采用同向啮合双螺杆挤压机(TwinLab-F 20/40,Brabender,德国)对物料进行挤压膨化,每次挤压膨化试验的固定喂料量为1 kg,喂料螺杆转速为80 r/min;试验前先将挤压机预热,设定机筒4个区域反应温度,喂料段温度为(50.0±0.1) ℃,混合段温度为(90.0±0.1) ℃,蒸煮段、熔融段和膨化模头温度为(120.0±0.1) ℃;待产品均匀挤出时开始取样。
本试验共分为9个组,分别为:对照组(CON组),豆粕;E组,挤压膨化豆粕;AO组,膨化前添加3%大豆毛油豆粕;CO组,膨化后添加3%大豆毛油豆粕;AO-CO组,膨化前后均添加3%大豆毛油豆粕;GE组,膨化前添加4%葡萄糖豆粕;GAO组,膨化前添加4%葡萄糖和3%大豆毛油豆粕;GCO组,膨化前添加4%葡萄糖和膨化后添加3%大豆毛油豆粕;GAO-CO组,膨化前添加4%葡萄糖和膨化前后均添加3%大豆毛油豆粕。试验样品在65 ℃下烘干48 h制成风干样,在空气中回潮后粉碎过1 mm分析筛。各组发酵底物主要营养水平见表2。
表2 各组发酵底物主要营养水平(干物质基础)

Table 2 Main nutrient levels of fermentation substrates in each group (DM basis)%

项目
Items		 组别 Groups
CON E AO CO AO-CO GE GAO GCO GAO-CO
干物质(烘干基础)
DM (drying basis)		 96.44 96.64 96.40 96.31 96.43 96.06 96.23 95.95 96.43
粗蛋白质 CP 45.99 45.37 43.90 43.54 42.64 43.23 42.98 42.34 41.87
粗脂肪 EE 1.99 1.63 4.66 4.57 7.30 1.42 2.12 4.35 5.54

1.2.2 试验动物及饲粮

本试验所用瘤胃液供体动物为中国农业大学房山肉牛试验基地的3头体重为600 kg、健康且装有永久性瘤胃瘘管的成年安格斯阉牛,每日于08:30和16:30进行饲喂,自由采食和饮水。瘘管牛的基础饲粮为参照NASEM(2016)[2]标准配制的全混合日粮(TMR),其组成及营养水平见表3。瘘管牛进行1周预饲,于第8天晨饲前采集瘤胃液,经4层纱布过滤后保存在39 ℃预热的保温壶中。
表3 基础饲粮组成及营养水平(干物质基础)

Table 3 Composition and nutrient levels of the basal diet (DM basis)%

原料 Ingredients 含量 Content 营养水平 Nutrient levels2) 含量 Content
全株玉米青贮 Whole-plant corn silage 27.39 粗蛋白质 CP 11.37
玉米秸秆 Corn straw 17.04 中性洗涤纤维 NDF 31.26
玉米 Corn 31.29 酸性洗涤纤维 ADF 19.33
豆粕 Soybean meal 9.60 钙 Ca 0.38
枣粉 Jujube powder 8.49 磷 P 0.32
预混料 Premix1) 2.45 代谢能 ME/(MJ/kg) 10.42
碳酸氢钠 NaHCO3 1.26 维持净能 NEm/(MJ/kg) 6.78
氯化钠 NaCl 1.26 增重净能 NEg/(MJ/kg) 4.31
磷酸氢钙 CaHPO4 1.22 总可消化养分 TDN 68.91
合计 Total 100.00

1)预混料为每千克饲粮提供 The premix provided the following per kg of the diet:VA 160 000 IU,VD 350 000 IU,VE 900 IU,VB 1 120 mg,烟酸 nicotinic acid 500 mg,Fe 1 200 mg,Cu (as copper sulfate) 150 mg,Zn (as zinc sulfate) 1 000 mg,Mn (as manganese sulfate) 500 mg。

2)代谢能、维持净能、增重净能和总可消化养分依据文献[19]计算,其余为实测值。ME, NEm, NEg and TDN were calculated based on reference [19], while the others were measured values.

1.3 检测指标及方法

1.3.1 样品养分测定

样品常规养分DM含量参照GB/T 6435—2014测定;粗蛋白质(CP)、中性洗涤不溶粗蛋白质(NDICP)、酸性洗涤不溶粗蛋白质(ADICP)和氮溶解指数(NSI)[20]含量参照GB/T 6432—2018,使用全自动凯氏定氮仪(KN580,阿尔瓦)测定;粗脂肪(EE)含量参照GB/T 6433—2006,使用全自动脂肪分析仪(XT15i,Ankom,美国)测定;中性洗涤纤维(NDF)和酸性洗涤纤维(ADF)含量参考Van Soest等[21]的方法,使用全自动纤维分析仪(Delta,Ankom,美国)测定;钙和磷含量分别参照GB/T 6436—2018和GB/T 6437—2018测定。
大豆毛油中长链脂肪酸组成测定:采用气相色谱仪(GC-2014,Shimadzu Corporation,日本)测定脂肪酸甲酯(FAME)含量,并计算脂肪酸含量[22]。

1.3.2 美拉德反应程度测定

挤压样品的颜色外观特征采用色差仪(CM-600D,柯尼卡美能达,日本)进行测定,使用白色标准比色板进行对比,每种样品测定5个重复,颜色指标使用样品亮度(L*)、红度(a*)、黄度(b*)和色差(ΔE)进行表征。
中间产物生成量和褐变程度测定:在Diftis等[23]的方法上稍作优化,称取1.0 g样品,加入到50 mL蒸馏水中,均质1 min,磁力搅拌2 h,确保样品溶解,离心20 min(离心机设置为3 800×g)后所得上清液经滤纸过滤后于4 ℃冰箱保存备用;取2.0 mL样品上清液用蒸馏水稀释2倍,涡旋混匀,然后以蒸馏水做空白,测定混合后液体在294和420 nm下的吸光度(OD)值并记录。

1.3.3 体外模拟瘤胃发酵

在培养瓶中(带几丁质胶塞,容积300 mL)称取3 g底物(干物质基础),每组样品5个重复。将150 mL在39 ℃预热后的缓冲液(pH=6.85)和75 mL过滤后的瘤胃液加入到培养瓶中;每个瓶子用氮气(N2)排净空气,立即用几丁质胶塞和螺旋盖密封,将瓶子放于恒温培养箱(HZQ-F160,豪诚)39 ℃培养16 h后取出,使用便携式pH计(Testo-205,Lenzkirch,德国)测定pH。为了测定DM消化率,采用尼龙袋(300目)过滤剩余发酵底物,清洗至水清[24];尼龙袋在65 ℃下烘干48 h至恒重;剩余物被用于进一步的CP检测分析。收集各瓶过滤后的消化液,检测MCP、氨态氮(NH3-N)和挥发性脂肪酸(VFA)含量,其中参考Makkar等[25]和Verdouw等[26]的方法测定MCP和NH3-N含量,采用气相色谱仪(GC-2014,Shimadzu Corporation,日本)测定上清液中VFA含量。

1.3.4 改良版体外三步法

根据Calsamiglia等[27]的研究,采用体外三步法评估样品中蛋白质的肠道消化情况。称取0.5 g 1.3.3中体外瘤胃降解后的残渣于纤维袋(ANKOM-FN57),封口后装入培养瓶中,每瓶最多放入30个纤维袋;每个培养瓶中加入2 L含有1 g/L胃蛋白酶(P-7000,Sigma)的pH=1.9的盐酸溶液,溶液提前预热;将培养瓶放在体外模拟发酵培养箱(Daisy Ⅱ,Ankom,美国),39 ℃旋转培养1 h;取出纤维袋并清洗干净,加入2 L预热的含有3 g/L胰蛋白酶和50 mg/L百里香酚的0.5 mol/L磷酸盐缓冲液,并将培养瓶放在体外模拟发酵培养箱(Daisy Ⅱ,Ankom,美国),39 ℃旋转培养24 h;取出纤维袋并清洗至水清,65 ℃烘箱内48 h烘至恒重,剩余为模拟小肠消化后的残渣样品,称重并测定CP含量。

1.4 相关计算公式

ΔE=[(ΔL*)2+(Δa*)2+(Δb*)2]1/2;

NSI(%)=100×可溶性氮含量/总氮含量;

RUP(g/kg)=CP(g/kg)×CP过瘤胃率(% CP);

小肠可消化粗蛋白质(IDCP,g/kg)=RUP(g/kg)×CP体外小肠消化率(%)+RDP(g/kg)×0.9×0.7;

养分过瘤胃率(%)=100×瘤胃16 h未降解残渣养分含量(g)/降解前袋内养分含量(g);

养分体外小肠消化率(%)=100×[瘤胃16 h未降解残渣养分含量(g)-酶培养后的残渣养分含量(g)/瘤胃16 h未降解残渣养分含量(g);

养分体外全消化道消化率(%)=100×[降解前袋内养分含量(g)-体外三步法后袋内未降解残渣养分含量(g)]/降解前袋内养分含量(g)。

式中:ΔL*表示亮度变化值;Δa*表示红度变化值;Δb*表示黄度变化值。

1.5 数据统计分析

所有结果数据首先在Excel 2016中进行处理,然后采用SPSS 21.0进行单因素方差分析,并采用Duncan氏法进行多重比较,结果采用平均值和均值标准误(SEM)表示,P<0.05为差异显著。

2 结果与分析

2.1 不同挤压膨化工艺对美拉德反应程度的影响

由表4可知,不同组之间样品美拉德反应程度相关指标均存在显著差异(P<0.05)。挤压膨化后,与CON组相比,各试验组样品a*、b*、ΔE和420 nm OD值均显著提高(P<0.05);除AO组外,其他试验组样品L*值均显著降低(P<0.05)。与E组相比,GE组样品a*、ΔE、294 nm OD和420 nm OD值显著提高(P<0.05);AO组样品L*、b*、294 nm OD和420 nm OD值显著提高(P<0.05),a*值显著降低(P<0.05)。此外,GAO组样品294 nm OD值显著低于只添加1种反应物的AO组和GE组(P<0.05),a*和ΔE值显著低于GE组(P<0.05)。
表4 挤压膨化工艺对美拉德反应程度的影响

Table 4 Effects of extrusion puffing processes on degree of Maillard reaction

项目
Items		 组别 Groups SEM P值
P-value
CON E AO CO AO-CO GE GAO GCO GAO-CO
亮度 L* 87.06a 85.25b 86.88a 80.70c 85.07b 77.42d 81.07c 74.98e 77.74d 0.64 <0.001
红度 a* 0.88i 4.71f 3.88h 6.08e 4.35g 8.15b 6.98d 8.57a 7.78c 0.35 <0.001
黄度 b* 16.25f 25.74d 26.39c 27.40b 28.11a 24.79e 25.51d 24.80e 25.71d 0.49 <0.001
色差 ΔE 20.79f 30.03e 29.73e 34.06c 32.13d 34.51bc 32.52d 36.26a 34.88b 0.66 <0.001
294 nm吸光度
OD294 nm		 1.335c 1.335c 1.693a 1.675a 1.392bc 1.622a 1.484b 1.640a 1.635a 0.026 <0.001
420 nm吸光度
OD420 nm		 0.113h 0.151f 0.352b 0.378a 0.301c 0.209e 0.203e 0.306c 0.272d 0.015 <0.001

同行数据肩标无字母或相同字母表示差异不显著(P>0.05),不同字母表示差异显著(P<0.05)。下表同。

In the same row, values with no letter or the same letter superscripts mean no significant difference (P>0.05), while with different letter superscripts mean significant difference (P<0.05). The same as below.

2.2 不同挤压膨化工艺对豆粕纤维和蛋白质组成的影响

在加热挤压膨化条件下,饲料中的蛋白质与添加的还原糖和油脂发生美拉德反应,改变了豆粕样品的纤维和蛋白质组成。由表5可知,在膨化前未添加葡萄糖处理的条件下,与CON组相比,E组、AO组、CO组和AO-CO组样品NDF和NDICP含量差异均不显著(P>0.05),但NSI均显著降低(P<0.05)。除GCO组外,GE组样品NDF和NDICP含量显著高于CON组和其他试验组(P<0.05),NSI显著低于CON组和膨化前未添加葡萄糖组(E组、AO组、CO组和AO-CO组)(P<0.05)。挤压膨化对各组样品ADF和ADICP含量无显著影响(P>0.05)。
表5 挤压膨化工艺对豆粕纤维和蛋白质组成的影响

Table 5 Effects of extrusion puffing processes on fiber and protein composition of soybean meal

项目
Items		 组别 Groups SEM P值
P-value
CON E AO CO AO-CO GE GAO GCO GAO-CO
中性洗涤纤维
NDF/%		 17.99bcd 15.44cd 14.62d 15.85cd 15.38cd 22.89a 18.44bc 20.45ab 18.69bc 0.50 <0.001
酸性洗涤纤维
ADF/%		 7.99 6.10 6.58 6.74 6.98 7.15 7.04 7.66 8.49 0.15 0.858
中性洗涤不溶
粗蛋白质
NDICP/% CP		 13.13bcd 9.33cd 7.48d 7.73cd 9.56cd 23.82a 14.37bc 18.28ab 16.77b 1.17 <0.001
酸性洗涤不溶
粗蛋白质
ADICP/% CP		 2.57 1.31 1.45 1.35 1.56 1.58 1.23 2.02 2.32 0.13 0.510
氮溶解指数
NSI/%		 13.65a 7.30b 6.22bc 6.61bc 6.48bc 4.93d 6.24bc 5.43cd 5.86cd 0.43 <0.001

2.3 不同挤压膨化工艺对豆粕在瘤胃和小肠降解的影响

由表6可知,与CON组相比,各试验组DM和CP的过瘤胃率、体外小肠消化率、体外全消化道消化率以及RUP和IDCP含量均显著提高(P<0.05)。GE组DM和CP的过瘤胃率以及RUP和IDCP含量(E组除外)显著高于其他试验组(P<0.05)。对于膨化后未进行大豆毛油包被处理,AO组DM和CP的过瘤胃率和体外小肠消化率以及RUP和IDCP含量显著低于E组(P<0.05);同样,GAO组DM和CP的过瘤胃率以及RUP和IDCP含量显著低于GE组(P<0.0)。对于膨化后进行大豆毛油包被处理,CO组和AO-CO组CP过瘤胃率分别显著高于E组和AO组(P<0.05)。但与上述结果相反的是,在膨化后包被大豆毛油后,GCO组DM和CP的过瘤胃率显著低于GE组(P<0.05);而GAO-CO组DM和CP的过瘤胃率与GAO组无显著差异(P>0.05),但GAO-CO组IDCP含量显著低于GAO组(P<0.05)。
表6 挤压膨化工艺对豆粕瘤胃和小肠降解的影响

Table 6 Effects of extrusion puffing processes on rumen and small intestine degradation of soybean meal

项目
Items		 组别 Groups SEM P值
P-value
CON E AO CO AO-CO GE GAO GCO GAO-CO
DM过瘤胃率
Rumen non-
degradable
rate of DM/%		 16.56e 34.73bc 24.55d 35.38bc 32.38c 42.63a 38.14b 36.49b 37.84b 1.18 <0.001
DM体外小肠消化率
Small intestinal
digestibility of
DM in vitro/%		 70.71e 90.55a 86.04d 89.45b 87.24c 90.38a 90.88a 91.16a 90.68a 1.21 <0.001
DM体外全消化
道消化率
Total digestive
tract digestibility of
DM in vitro/%		 95.15e 96.72ab 96.57ab 96.27c 95.87d 95.90d 96.52ab 96.77a 96.47bc 0.10 <0.001
CP过瘤胃率
Rumen non-
degradable rate
of CP/%		 17.84f 52.84d 37.07e 58.00bc 53.33cd 70.23a 62.64b 59.90b 63.02b 2.33 <0.001
瘤胃不可降
解蛋白质
RUP/(g/kg)		 79.12f 231.71cd 156.86e 243.22bc 219.27d 291.66a 259.08b 243.33bc 254.42b 9.36 <0.001
CP体外小肠消化率
Small intestinal
digestibility of CP
in vitro/%		 96.36e 99.37a 98.97d 99.31a 99.14c 99.34a 99.37a 99.35a 99.23b 0.18 0.002
小肠可消化
粗蛋白质
IDCP/(g/kg)		 305.82h 360.52ab 323.02g 352.52cd 338.29f 367.64a 354.79bc 344.36ef 346.50de 2.84 <0.001
CP体外全消化
道消化率
Total digestive tract
digestibility of CP
in vitro/%		 99.30d 99.67a 99.64a 99.61ab 99.53bc 99.53bc 99.59abc 99.61ab 99.50c 0.02 0.009

2.4 不同挤压膨化工艺下豆粕瘤胃发酵参数的影响

由表7可知,体外发酵16 h后,各试验组瘤胃液pH和NH3-N含量与CON组相比显著降低(P<0.05),GAO-CO组MCP含量显著低于AO-CO组(P<0.05)。对于VFA而言,加热挤压膨化处理没有对总挥发性脂肪酸(TVFA)含量产生显著影响(P>0.05),但是改变了瘤胃液VFA的组成,加热挤压膨化后,与CON组相比,各试验组瘤胃液中异丁酸、异戊酸和总支链挥发性脂肪酸(TBCVFA)含量显著降低(P<0.05)。在膨化前未添加葡萄糖的试验组中,CO组和AO-CO组乙酸/丙酸值显著低于E组和AO组(P<0.05);但在膨化前添加葡萄糖的试验组中,与GE组相比,GAO组、GCO组和GAO-CO组丙酸含量显著降低(P<0.05),乙酸/丙酸值显著提高(P<0.05)。AO组戊酸显著高于CON组和GE组(P<0.05)。此外,在膨化前未添加葡萄糖的试验组中,膨化后进行大豆毛油包被处理后,AO-CO组丁酸含量显著低于AO组(P<0.05);但在膨化前添加葡萄糖的试验组中,膨化后进行大豆毛油包被处理后,GCO组和GAO-CO组丁酸含量显著高于GE组和GAO组(P<0.05)。
表7 挤压膨化工艺对豆粕瘤胃发酵参数的影响

Table 7 Effects of extrusion puffing processes on rumen fermentation parameters of soybean meal

项目
Items		 组别 Groups SEM P值
P-value
CON E AO CO AO-CO GE GAO GCO GAO-CO
pH 6.61a 6.57bc 6.58b 6.51e 6.52de 6.53de 6.53de 6.54cde 6.56bcd 0.01 <0.001
氨态氮
NH3-N/(mg/dL)		 49.27a 34.45bc 38.80b 28.12cde 30.48cd 19.22f 22.70ef 24.46df 25.20df 1.45 <0.001
微生物蛋白
MCP/(mg/mL)		 0.74ab 0.75ab 0.71ab 0.78ab 0.80a 0.77ab 0.67ab 0.64ab 0.59b 0.02 0.012
乙酸
Acetic acid/
(mmol/L)		 33.68b 38.84a 38.90a 37.18ab 33.74b 36.56ab 37.08ab 39.71a 38.19a 0.47 0.005
丙酸
Propionic acid/
(mmol/L)		 14.74ab 14.22ab 14.28ab 15.31a 15.57a 15.17a 13.65bc 13.29bc 12.38c 0.20 0.003
异丁酸
Isobutyric acid/
(mmol/L)		 0.69a 0.42c 0.56b 0.36d 0.43c 0.34d 0.36d 0.41c 0.41c 0.02 <0.001
丁酸
Butyric acid/
(mmol/L)		 5.01a 4.88a 5.06a 4.66ab 4.23b 4.24b 4.28b 4.93a 5.09a 0.08 0.006
异戊酸
Isovaleric acid/
(mmol/L)		 2.01a 1.02c 1.43b 0.85de 0.98c 0.72f 0.81ef 0.94cd 0.95cd 0.06 <0.001
戊酸
Valeric acid/
(mmol/L)		 0.33c 0.52ab 0.63a 0.44abc 0.38bc 0.37c 0.42abc 0.48abc 0.48abc 0.02 <0.001
乙酸/丙酸
Acetic acid/
propionic acid		 2.28cd 2.73b 2.74b 2.27cd 2.17d 2.41c 2.73b 2.99a 3.09a 0.05 <0.001
总支链挥发性
脂肪酸
TBCVFA/
(mmol/L)		 2.70a 1.44c 2.00b 1.22de 1.40c 1.05f 1.17ef 1.34cd 1.36cd 0.08 <0.001
总挥发性脂肪酸
TVFA/(mmol/L)		 56.34 60.03 60.74 58.96 55.40 57.39 56.79 59.64 57.54 0.57 0.339

3 讨论

3.1 不同挤压膨化工艺对美拉德反应程度的影响

美拉德反应是广泛存在于饲料加工和储存过程中的非酶促褐变反应,通过对饲料样品的颜色进行测定与分析,能在一定程度上反映样品美拉德反应程度大小[11]。本研究中,各试验组豆粕在挤压膨化后,ΔE值显著增加,说明加工过程中发生了美拉德反应;在膨化后未进行大豆毛油包被的各试验组中,膨化前添加葡萄糖的GE组和GAO组的ΔE值显著高于膨化前未添加葡萄糖的E组和AO组,说明添加葡萄糖后,挤出物在机筒内挤压膨化过程中的美拉德反应程度增大,表明葡萄糖促进了美拉德反应的发生,这与张恺容[11]的研究结果一致。但是在膨化后未进行大豆毛油包被的各试验组中,AO组相较于E组以及GAO组相较于GE组L*和b*值显著提高,a*值显著降低,且GAO组ΔE值显著低于GE组,表明在挤压膨化过程中添加不饱和脂肪酸含量较高的大豆毛油可能会抑制美拉德反应。这与Chen等[28]的研究结果一致,在高水分豌豆蛋白质挤压膨化过程中,添加不饱和脂肪酸亚油酸组挤出物相对于未添加脂肪酸组挤出物的L*值更高,a*值更低。研究发现,脂肪酸的融化能够促进光的反射[29],使样品外观更加光泽,这与L*值的提高直接相关。此外,脂肪酸小液滴除了发挥稀释挤出物的美拉德反应褐变产物颜色作用外,还可能阻止了有色大分子的形成[30]。在美拉德反应过程中,环化、脱水、缩合等多种反应最终导致棕色的含氮聚合物以及共聚物类黑精(MeH)的生成[31],MeH的生成量与美拉德反应程度成正比,在420 nm处有最大OD值,可用此值表征褐变程度[32-33];同时,美拉德反应中间产物在290 nm波长下具有特征性吸收[34]。因此,使用酶标仪测定294和420 nm下OD值能够反映美拉德反应的中间产物生成量和褐变程度,OD值越大,表明中间产物生成量越多,褐变程度越大,美拉德反应程度越大[35]。本研究发现,对于未包被的各组,GE组294和420 nm OD值显著高于CON组,但是GAO组294 nm OD值相较于只添加1种反应物的AO组和GE组显著降低,这进一步说明不饱和脂肪酸的添加阻止了有色大分子的形成,导致挤压过程中葡萄糖和饲料氨基酸的美拉德反应程度降低。而包被大豆毛油后,各试验组294和420 nm OD值显著提高,这主要与包被的大豆毛油其本身对光的吸收有关。

3.2 不同挤压膨化工艺对豆粕纤维和蛋白质组成的影响

挤压膨化技术集成了原料的混合、剪切、熔化以及成型等多个单元操作[36],在高温、高压和高剪切力的协同作用下,可以改变样品营养物质的分子结构[37]。本研究发现,GE组NDF和NDICP含量相较于CON组显著提高,这与葡萄糖的添加密切相关。挤压膨化技术对饲料原料纤维和蛋白质水平的影响与饲料原料的化学组成密切相关,还原糖在高温、高压和高剪切力的协同作用下与植物蛋白质游离的氨基发生美拉德反应,通过共价相互作用形成蛋白质-糖结合物,随着加热的继续,结合物最终会形成棕色大分子聚合物,从而改变植物蛋白质的结构和理化性质[38]。但是,当在膨化前添加富含不饱和脂肪酸的大豆毛油后,GAO组和GAO-CO组NDICP含量相较于GE组显著降低,这可能是因为脂质在挤压膨化的过程中扮演着润滑剂、表面活性剂和乳化剂等角色,可以增加蛋白质的分子流动性,降低熔体黏度,从而影响聚合反应和纤维取向[39-40]。Chen等[28]研究发现,高不饱和度的脂肪酸增加了高机械能量消耗,不利于挤出物纤维结构的形成。在挤压过程中,温度是导致蛋白质结构变化的主要因素之一,当挤压温度达到植物蛋白质的变性温度时,会导致其天然结构的破坏[41]。本研究中,与CON组相比,各试验组经过120 ℃挤压膨化后,NSI显著降低,说明饲料蛋白质在高温高压作用下发生氢键断裂,二级、三级和四级结构展开,暴露内部巯基和疏水残基[42],然后在熔融区当挤压温度达到最大值时,通过巯基(-SH-)-二硫键(-S-S-)交换反应形成分子间二硫键[43]。进一步研究发现,较高的挤压温度(>110 ℃)可能会打破新形成的二硫键[44]。由于二硫键的共价相互作用,形成了少量的低聚物[45]。当低聚物缔合浓度超过临界值时,它们开始聚集,导致不溶性团聚体的形成,降低了挤压蛋白的溶解度[12,44-45]。Tian等[46]研究发现,糖基化的大豆分离蛋白质在酸性条件下溶解性较好,这与本研究中ADF和ADICP在挤压膨化后没有显著变化的结果相一致。

3.3 不同挤压膨化工艺对豆粕在瘤胃和小肠降解的影响

在挤压膨化过程中,还原糖与饲料中的蛋白质发生不可逆的美拉德反应,挤压产物能够降低反刍动物瘤胃中蛋白质的消化率,从而提高豆粕优质蛋白质的过瘤胃效率[2]。本研究发现,与CON组相比,各试验组DM和CP的过瘤胃率、体外小肠消化率、体外全消化道消化率以及RUP和IDCP含量均显著提高,这与NASEM(2016)结果[2]一致,其机制主要是饲料蛋白质在机筒高温、高压和高剪切力的作用下,形成不溶性的团聚体[42],降低了NSI,进而提高了过瘤胃率;同时,还原糖的添加与植物蛋白质游离的氨基发生美拉德反应,通过共价相互作用形成蛋白质-糖结合物[38],GE组DM和CP的过瘤胃率以及RUP和IDCP含量显著高于其他试验组,这与样品美拉德反应程度以及纤维和蛋白质组成变化的结果相一致。但是膨化前添加大豆毛油后,高度不饱和脂肪酸作为一种润滑剂,能够增加蛋白质的分子流动性,影响在机筒内发生的美拉德反应和聚合反应程度,最终影响挤出物的纤维取向[28,39],对饲料蛋白质过瘤胃保护效果产生了负面影响。对于膨化后大豆毛油包被处理的各试验组,膨化前未添加葡萄糖,膨化后包被大豆毛油显著提高了DM过瘤胃率和RUP含量,这与油脂对饲料颗粒的包被作用有关[47]。但是膨化前添加葡萄糖各组包被大豆毛油后没有显著提高过瘤胃效果,这与现有的研究结果并不一致。GE组样品在膨化后蛋白质和纤维结构发生改变,瘤胃微生物难以消化,而低浓度油脂的包被为瘤胃微生物供能提供发酵底物[18],促进了瘤胃微生物对饲料的消化。饲料样品在经过反刍动物瘤胃微生物消化后,进入到后肠道参与酶消化过程。研究发现,挤压膨化增加了蛋白质的表面积[48],使其更易与蛋白酶结合,从而提高CP的消化率,并且糖基化的大豆分离蛋白质在酸性条件下溶解性较好[46,49],这与本研究中各试验组挤压膨化没有显著改变饲料蛋白质的ADICP含量,并且DM和CP的体外小肠消化率显著高于CON组的结果相一致。
体外瘤胃发酵16 h后,各组瘤胃液pH均超过6,表明瘤胃体外发酵环境稳定[50]。饲料中的蛋白质在瘤胃中分解会产生肽和氨基酸,它们参与细菌的构成或者脱氨基形成氨(NH3)、二氧化碳(CO2)和VFA。支链氨基酸进一步分解产生支链挥发性脂肪酸(异丁酸和异戊酸)[2]。本研究中,挤压膨化处理没有显著改变TVFA含量,但是各试验组中,瘤胃液的pH以及NH3-N、异丁酸、异戊酸和TBCVFA含量相较于CON组显著降低。这是因为饲料蛋白质在挤压膨化高温高压的作用下形成不溶性的团聚体,降低了NSI,导致蛋白质的瘤胃消化率降低。同时,在膨化前添加还原糖后,还原糖通过美拉德反应形成蛋白质-糖结合物,饲料蛋白质中NDICP含量显著增加,瘤胃微生物难以消化利用蛋白质,进一步降低了蛋白质的瘤胃消化率。蛋白质消化率降低,导致瘤胃中合成的NH3-N和支链挥发性脂肪酸减少[14],从而降低了瘤胃液的pH。

4 结论

本研究采用双螺杆挤压膨化的方法处理豆粕,研究了葡萄糖、大豆毛油添加以及大豆毛油包被等对挤出物美拉德反应程度、纤维和蛋白质组成、瘤胃发酵和小肠消化情况的影响。结果表明,葡萄糖的添加增加了挤压膨化过程中美拉德反应的程度,促进了豆粕优质蛋白质的过瘤胃和小肠消化吸收。同时,膨化前添加富含不饱和脂肪酸的大豆毛油会抑制挤压膨化过程中美拉德反应的程度。因此,挤压膨化过程中,添加还原糖能够提高膨化效果,进一步提高优质蛋白质的过瘤胃效率,但是不应添加富含不饱和脂肪酸的大豆毛油。

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