Chinese Journal of Animal Nutrition >

2025 , Vol. 37 >Issue 11: 7941 - 7954

DOI: https://doi.org/10.12418/CJAN2025.646

RESEARCH PAPER

Research on Removal Technology of Tea Saponin in Camellia Seed Meal

  • WANG Li , 1, 2 ,
  • ZHAO Chen 1 ,
  • HE Beibei 1, 2 ,
  • DUAN Tao 1, 2 ,
  • SHI Jingjing 1, 2 ,
  • MO Yanting 1 ,
  • WANG Yongwei , 1, 2, * ,
  • WANG Weiwei , 1, 2, *
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  • 1 Academy of National Food and Strategic Reserves Administration, Beijing 100037, China
  • 2 Key Laboratory of Grain and Oil Biotechnology, Academy of National Food and Strategic Reserves Administration, Beijing 100037, China
*WANG Yongwei, associate professor, E-mail: ;
WANG Weiwei, professor, E-mail:

Received date: 2025-03-28

  Online published: 2025-11-14

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Abstract

This study was conducted to investigate the effects of different removal technologies on tea saponin content and nutritional values of camellia seed meal before and after treatment, with the aim of obtaining the best technology and optimizing the combination of strains and the co-fermentation process of bacteria and enzymes. Firstly, the effects of different hot water extraction time, material to water ratios and extraction temperatures on the removal of tea saponin were explored, and the optimal hot water extraction conditions were screened out; secondly, taking the removal rate of tea saponin as an indicator, the best combination of fermentation strains and the best process were screened out, and taking the degradation rate of crude fiber as an indicator, the best ratio of enzyme preparations was screened out; finally, taking the removal rate of tea saponin as an indicator, the best co-fermentation process of bacteria and enzymes was screened out. The results showed as follows: 1) the optimal hot water extraction conditions obtained through screening were extraction time of 4 hours, material to water ratio of 1∶11, and extraction temperature of 80 ℃. The removal rate of tea saponin was 72.75%. However, after extraction, the contents of crude protein, total sugar and reducing sugar in camellia seed meal were significantly reduced (P<0.05), and the crude fiber content was significantly increased (P<0.05). 2) Three strains of tea saponin-degrading bacteria were screened out, which were Saccharomyces CJM27, Bacillus subtilis CYB41 and Aspergillus niger CMCC. 3) The optimal process for the co-fermentation of bacteria and enzymes involved: first, 150 U/g cellulase, 50 U/g xylanase and 100 U/g β-glucanase were added, and enzymatic hydrolysis was carried out for 2 days under the condition of material to water ratio of 1.0∶0.4; then 1×106 CFU/mL Lactobacillus casei A20170701, 1×106 CFU/mL Saccharomyces CJM27, 1×108 CFU/mL Bacillus subtilis CYB41 and 1×107 CFU/mL Aspergillus niger CMCC were inoculated, and the material to water ratio was adjusted to 1.00∶0.50; after fermentation in a constant temperature incubator at 32 ℃ for 84 hours, the removal rate of tea saponin reached 61.64%. After the co-fermentation of bacteria and enzymes, the contents of crude protein, reducing sugar and total amino acids in camellia seed meal were increased by 7.80%, 168.32% and 7.97% (P<0.05), while the contents of crude fiber and total phenol were decreased by 13.70% and 11.64% (P<0.05), respectively. In conclusion, the co-fermentation of bacteria and enzymes can effectively reduce the tea saponin content in camellia seed meal and enhance its nutritional values. The effect is superior to that of hot water extraction, which provides effective technical support for the development and utilization of camellia seed meal.

Key words： camellia seed meal; tea saponin; probiotics; enzyme

Cite this article

WANG Li , ZHAO Chen , HE Beibei , DUAN Tao , SHI Jingjing , MO Yanting , WANG Yongwei , WANG Weiwei . Research on Removal Technology of Tea Saponin in Camellia Seed Meal[J]. Chinese Journal of Animal Nutrition, 2025 , 37(11) : 7941 -7954 . DOI: 10.12418/CJAN2025.646

我国是世界上油茶分布最广、品种最多的国家,2020年我国油茶种植面积约7 175万亩(1亩≈666.7 m2)。据国家林业和草原局对茶油产业的规划,到2025年,我国油茶种植面积将扩大2 000万亩。同时,2020年我国油茶籽产量为314.16万t,榨油后剩余的油茶籽粕产量达250万t。油茶籽粕不仅富含粗蛋白质、粗脂肪和粗纤维等营养成分,而且含有糖类、多酚和茶皂素等生物活性成分[1],是潜在的饲料资源。茶皂素是一类糖苷类化合物,具有抑菌和抗氧化等作用[2]。适量的茶皂素具有促进畜禽生长发育[3]、调节免疫力[4]和抗氧化能力[5]、减少腹泻[6]以及保护肝脏[7]等作用。但是,高剂量的茶皂素具有溶血作用,会导致畜禽出现胃中毒、肝脏损伤[8]、惊厥昏迷甚至死亡的现象,尤其是对鱼等水产动物和昆虫,因此常被用作清塘剂和杀虫剂使用[9]。
油茶籽粕中茶皂素脱毒的常见方法有热水浸提法、有机溶剂浸提法、微生物发酵法和酶解法等[10]。其中,热水浸提法虽可有效去除抗营养因子,茶皂素脱除率为31.2%~95.5%[11-12],但是会产生大量废水;有机溶剂提取法虽可实现高效提取茶皂素,但存在有机试剂残留和营养物质损失的问题;微生物发酵油茶籽粕,不仅可以使茶皂素脱除率为34.06%~94.91%[13-14],还可以提高油茶籽粕的营养物质消化率,产生维生素、有机酸和促生长因子等物质[15]。油茶籽粕未进行脱毒处理时,肉鸡饲粮中添加1%即可对肉鸡生长性能产生负面影响;而脱毒处理后的油茶籽粕在肉鸡饲粮中可替代15%豆粕[16],部分优质脱毒油茶籽粕在猪饲粮中添加量可达15%[17]。
虽然热水浸提和生物降解茶皂素的方法均已有报道,但是这2种方法处理油茶籽粕后的营养价值变化对比却鲜有报道,且利用菌酶协同技术处理油茶籽粕的报道不多,因此本研究旨在考察不同脱除工艺对油茶籽粕处理前后茶皂素含量及其营养价值的影响,以期获得最佳处理工艺,并优化出最佳菌种组合和菌酶协同发酵工艺,为油茶籽粕的开发和利用提供参考。

1 材料与方法

1.1 试验材料

油茶籽粕为市购产品。芽孢杆菌、酵母菌、乳酸菌和黑曲霉均由国家粮食和物资储备局科学研究院粮食品质营养研究所筛选并保存。LB培养基、MRS培养基和PTB培养基购自北京奥博星生物技术有限责任公司。茶皂素无机盐固体培养基配制:0.25 g/L K2HPO4,0.25 g/L MgSO4·H2O,0.5 g/L KCl,0.5 g/L (NH4)2SO4,0.005 g/L CaCl2,0.003 g/L FeCl3·6H2O,0.003 g/L ZnSO4,0.001 g/L CoCl2,0.001 g/L MnSO4,0.001 g/L CuSO4,pH 6.3~6.5,每100 mL加入1 g茶皂素粗提物。茶皂素粗提物(纯度为98%)购自北京索莱宝科技有限公司。

1.2 试验方法

1.2.1 茶皂素含量的测定

采用香草醛-浓硫酸比色法[18],分别配制成0.002、0.004、0.006、0.008和0.010 mg/mL质量浓度的茶皂素标准溶液,于550 nm处测定吸光度值。以吸光度值为Y轴,茶皂素含量为X轴,绘制标准曲线。称取5 g油茶籽粕,加入50 mL 75%乙醇超声提取40 min,离心,上清液定容至50 mL,吸取0.1 mL样品提取液至50 mL离心管中,后处理方法同标准品。根据标准曲线,计算茶皂素含量,计算公式为:
X=[(C×V×K)/m]×100。
式中:X为油茶籽粕中茶皂素含量(%);C为油茶籽粕中茶皂素质量浓度(mg/mL);V为提取液体积(mL);K为稀释倍数;m为称样质量(g)。

1.2.2 热水浸提茶皂素单因素及正交试验

准确称取100 g油茶籽粕,通过改变料水比[1∶8、1∶9、1∶10、1∶11和1∶12(g∶mL)]、浸提时间(2、3、4、5和6 h)或浸提温度(70、75、80、85和90 ℃)进行单因素试验。由单因素试验结果,选取3个梯度,以料水比、浸提时间和浸提温度为考察因素,设计L9(33)正交试验(表1),确定最佳热水浸提工艺。测定粗蛋白质、粗纤维、茶皂素、总糖和还原糖含量,计算茶皂素脱除率和常规营养成分降解率。
表1 热水浸提茶皂素正交试验因素水平

Table 1 Factors and levels of orthogonal experiment for hot water extraction of tea saponin

水平
Levels		 浸提时间
Extraction time (A)/h		 料水比
Material to water ratio (B)/(g∶mL)		 浸提温度
Extraction temperature (C)/℃
1 2 1∶9 80
2 3 1∶10 85
3 4 1∶11 90

1.2.3 发酵菌株的筛选

分别配制茶皂素浓度为1.5%和2.5%的茶皂素无机盐固体培养基,将16株芽孢杆菌、10株酵母菌和3株黑曲霉在固体培养基上分别划线,筛选长势较好的菌株作为目标菌株;乳酸菌选用干酪乳杆菌A20170701。

1.2.4 单菌固态发酵试验

称取油茶籽粕样品100 g于烧杯中,酵母菌、芽孢杆菌和干酪乳杆菌培养16 h接种,黑曲霉培养5 d接种,按照原料中活菌数为5×107 CFU/g的接种量分别接种,并按照料水比为1.0∶0.4(mg∶mL)补齐水分,搅拌均匀后用4层纱布封口,放置到生化培养箱中培养3 d,酵母菌、芽孢杆菌和干酪乳杆菌固体发酵培养温度为37 ℃,黑曲霉培养温度为28 ℃,期间每12 h搅拌1次,每组3个重复。

1.2.5 多菌株混合发酵工艺筛选

发酵菌株选用2菌株、3菌株或4菌株进行混合发酵,发酵工艺分为3种不同处理:好氧发酵3 d、厌氧发酵3 d以及纤维素酶解2 d+厌氧发酵3 d,每组3个重复。好氧发酵用4层纱布封口,厌氧发酵用4层纱布外加1层保鲜膜封口,纤维素酶添加量为100 U/g。

1.2.6 多菌株接种量正交试验

以上述筛选出来的最佳复合菌株发酵工艺为基础,以各菌株为因素,接种量为水平,设计4因素3水平L9(34)正交试验(表2)。
表2 多菌株接种量正交试验因素水平

Table 2 Factors and levels of orthogonal experiment for inoculation volume of multiple strainsCFU/mL

水平
Levels		 干酪乳杆菌A20170701
Lactobacillus casei
A20170701 (A)		 酵母菌CJM27
Saccharomyces
CJM27 (B)		 枯草芽孢杆菌CYB41
Bacillus subtilis
CYB41 (C)		 黑曲霉CMCC
Aspergillus niger
CMCC (D)
1 1×106 1×106 1×106 1×106
2 1×107 1×107 1×107 1×107
3 1×108 1×108 1×108 1×108

1.2.7 不同酶制剂对纤维素的降解效果

采用纤维素酶、木聚糖酶和β-葡聚糖酶3种酶以单酶、双酶和三酶的形式进行固态发酵,酶解时间为24 h,酶解温度为37 ℃,料水比1.0∶0.4(mg∶mL),每种酶添加量均为100 U/g。确定最佳酶组合之后,在总酶添加量300 U/g的基础上,进行各酶之间不同添加比例的试验,测定样品粗纤维和还原糖含量。

1.2.8 油茶籽粕菌酶协同固态发酵

选用1.2.7中筛选的最佳酶解工艺酶解2 d,然后按照1.2.6中筛选的工艺接种菌株,进行不同料水比[1.00∶0.40、1.00∶0.45、1.00∶0.50、1.00∶0.55和1.00∶0.60(mg∶mL)]、不同发酵时间(60、72、84、96和108 h)和不同发酵温度(30、32、34、37和40 ℃)的单因素试验。根据单因素试验结果,确定3个因素的3个水平,进行L9(33)正交试验(表3)。
表3 菌酶协同发酵正交试验因素水平

Table 3 Factors and levels of orthogonal experiment co-fermentation of bacteria and enzymes

水平
Levels		 料水比
Material to water ratio (A)/(mg∶mL)		 发酵时间
Fermentation time (B)/h		 发酵温度
Fermentation temperature (C)/℃
1 1.00∶0.50 84 32
2 1.00∶0.55 96 34
3 1.00∶0.60 108 37

1.2.9 常规营养成分含量测定

粗蛋白质含量采用FOSS 8400凯氏定氮仪进行测定,粗纤维采用ANKOM 2000i全自动纤维分析仪进行测定,粗脂肪含量采用FOSS Soxtec8000脂肪测定仪进行测定,氨基酸含量采用曼默博尔A300 Advanced氨基酸分析仪进行测定。

1.2.10 样品中还原糖含量的测定

3,5-二硝基水杨酸(DNS)试剂配制:称取6.9 g结晶重蒸馏酚溶于15.2 mL 10%氢氧化钠溶液中,稀释至69 mL后加入6.9 g亚硫酸氢钠,作为A液;将255 g酒石酸钾钠加入300 mL 10%氢氧化钠溶液中,再加入880 mL 1% DNS溶液,作为B液;将A液和B液混合,得黄色溶液,贮于棕色瓶中备用,常温下放置1周后使用。采用无水D-葡萄糖绘制标准曲线。
精密称取0.1 g样品,置于具塞锥形瓶中,加入蒸馏水25 mL,超声处理20 min,取出后放至室温,摇匀,过滤;取2 mL溶液,置于25 mL具塞大试管中,空白对照加2 mL蒸馏水,样品加入2 mL DNS试剂,摇匀,90 ℃水浴显色6 min,冷却至室温,加蒸馏水定容至25 mL,摇匀,于540 nm处测定吸光度值。

1.3 数据统计分析

试验结果数据采用SPSS 22.0软件进行统计分析,油茶籽粕处理前后营养成分差异显著性比较采用独立样本t检验,其他数据采用单因素方差分析和Duncan氏法多重比较,结果数据采用“平均值±标准误”形式表示,P<0.05表示差异显著。

2 结果与分析

2.1 热水浸提茶皂素单因素及正交试验

热水浸提茶皂素单因素试验结果显示(图1),当浸提时间为3 h时,茶皂素脱除率最高,为49.28%;当料水比为1∶10时,茶皂素脱除率最高,为54.16%;当浸提温度为85 ℃时,茶皂素脱除率最高,为61.82%。正交试验结果见表4,由极差(R)值可以看出,对油茶籽粕茶皂素脱除率影响的主次因素依次为:浸提温度(C)>浸提时间(A)>料水比(B)。由表5可知,浸提时间和浸提温度对茶皂素脱除率具有显著影响(P<0.05),综合方差齐性检验显示,最佳浸提工艺组合为A3B3C1,即热水浸提时间为4 h、料水比为1∶11、浸提温度为80 ℃。采用最佳工艺进行热水浸提,由表6可知,油茶籽粕茶皂素脱除率为72.75%;同时,与处理前相比,处理后油茶籽粕粗蛋白质、总糖和还原糖含量分别显著降低了20.71%、38.29%和65.63%(P<0.05),粗纤维含量显著提高了25.93%(P<0.05)。
图1 不同浸提时间、料水比和浸提温度对茶皂素脱除率的影响

数据点标注不同小写字母表示差异显著(P<0.05)。图2同。

Fig.1 Effects of different extraction time, material to water ratios and extraction temperatures on removal rate of tea saponin

Data points with different small letters mean significant difference (P<0.05). The same as Fig.2.

表4 热水浸提茶皂素正交试验结果

Table 4 Results of orthogonal experiment for hot water extraction of tea saponin

项目
Items		 浸提时间
Extraction
time (A)		 料水比
Material to water
ratio (B)		 浸提温度
Extraction
temperature (C)		 茶皂素脱除率
Removal rate of
tea saponin/%
1 1 2 3 68.31±0.10
2 1 3 2 68.66±0.38
3 1 1 1 69.28±0.21
4 2 3 3 65.73±0.95
5 2 2 1 69.30±0.05
6 2 1 2 68.11±0.22
7 3 3 1 71.75±0.47
8 3 1 3 66.94±0.18
9 3 2 2 68.13±1.62
K1 206.25 204.33 210.33
K2 203.14 205.74 204.90
K3 206.82 206.14 200.98
k1 68.75 68.11 70.11
k2 67.71 68.58 68.30
k3 68.94 68.71 66.99
R 1.23 0.60 3.12
主次因素Primary and secondary factors C>A>B
最优组合Optimal combination A3B3C1

表中“K”为数列中不同水平对应茶皂素脱除率的和,“k”为“K”的平均值,“R”为极差,即k最大值-k最小值。表9和表12同。

In the table, “K” represented the sum of removal rates of tea saponin corresponding to different levels in the sequence, “k” was the average value of “K”, and “R” was the range, which was the maximum value of k minus the minimum value of k. The same as Table 9 and Table 12.

表5 热水浸提茶皂素正交试验方差分析

Table 5 Analysis of variance of orthogonal experiment for hot water extraction of tea saponin

项目
Items		 离差平方和
Sum of squares of
deviations		 自由度
Degree of
freedom		 均方
Mean square		 F值
F-value		 P值
P-value
浸提时间Extraction time (A) 7.855 2 3.928 3.722 0.042
料水比Material to water ratio (B) 1.807 2 0.903 0.856 0.440
浸提温度Extraction temperature (C) 43.928 2 21.964 20.817 <0.001
误差Error 21.102 20 1.055
表6 热水浸提前后油茶籽粕成分变化

Table 6 Composition changes of camellia seed meal before and after hot water extraction

项目
Items		 处理前
Before processing		 处理后
After processing
粗蛋白质Crude protein/% 7.05±0.04a 5.59±0.06b
粗纤维Crude fiber/% 38.61±0.45b 48.62±0.06a
茶皂素Tea saponin/% 15.30±0.01a 4.17±0.01b
总糖Total sugar/(mg/g) 19.35±0.03a 11.94±0.02b
还原糖Reducing sugar/(mg/g) 9.69±0.02a 3.33±0.14b

同行数据肩标不同字母表示差异显著(P<0.05),相同字母或无字母表示差异不显著(P>0.05)。表14同。

In the same row, values with different letter superscripts means significant difference (P<0.05), while with the same letter or no letter superscripts means no significant difference (P>0.05). The same as Table 14.

2.2 发酵菌株的筛选

采用1.5%茶皂素培养基筛选出2株可培养的酵母菌(FJM13和CJM27)和3株长势较好的枯草芽孢杆菌(PYB1、CYB41和CYB5-1);另外,2株黑曲霉(16404和CMCC)在2.5%茶皂素培养基中长势均较好。采用筛选出的7种单菌进行油茶籽粕单菌发酵,结果(表7)显示,酵母菌CJM27、枯草芽孢杆菌CYB41和黑曲霉CMCC处理茶皂素脱除率最高,分别达到36.68%、29.49%和31.84%。
表7 不同菌株对油茶籽粕茶皂素的脱除效果

Table 7 Removal efficiency of tea saponin from camellia seed meal by different microbial strains%

项目
Items		 菌株编号
Strain No.		 茶皂素含量
Tea saponin
content		 茶皂素脱除率
Removal rate of
tea saponin

酵母菌Saccharomyces		 FJM13 10.84±0.11bc 29.16±0.72cd
CJM27 9.69±0.09e 36.68±0.59a


枯草芽孢杆菌Bacillus subtilis		 PYB1 13.00±0.04a 15.04±0.26e
CYB41 10.89±0.15c 29.49±0.35c
CYB5-1 10.91±0.21bc 28.75±1.31cd

黑曲霉Aspergillus niger		 16404 11.09±0.30b 27.55±1.99d
CMCC 10.43±0.06d 31.84±0.39b

同列数据肩标不同字母表示差异显著(P<0.05),相同字母或无字母表示差异不显著(P>0.05)。表8和表11同。

In the same column, values with different letter superscripts means significant difference (P<0.05), while with the same letter or no letter superscripts means no significant difference (P>0.05). The same as Table 8 and Table 11.

2.3 多菌株混合发酵工艺筛选

干酪乳杆菌能分泌多种酶类,在发酵过程中可以产生一些多糖、有机酸等益生物质,并可以降低物料pH,延长物料保存期,因此添加干酪乳杆菌为混合发酵菌株。由表8可知,在2菌株组合固态发酵中,好氧发酵3 d,茶皂素脱除率均较低,其中CYB41+CMCC处理脱除率显著高于其他处理(P<0.05);在3菌株和4菌株组合中,先酶解2 d再用A20170701+CJM27+CYB41+CMCC处理厌氧发酵3 d茶皂素脱除率最高,脱除率为45.00%。
表8 多菌株混合发酵工艺筛选

Table 8 Screening of multi-strain mixed fermentation processes%

项目
Items		 发酵方式
Fermentation method		 茶皂素脱除率
Removal rate of tea saponin
A20170701+CJM27 好氧3 d 13.45±0.17d
A20170701+CYB41 好氧3 d 15.11±0.14c
A20170701+CMCC 好氧3 d 17.58±0.17b
CJM27+CMCC 好氧3 d 15.68±0.14c
CJM27+CYB41 好氧3 d 15.39±0.04c
CYB41+CMCC 好氧3 d 19.93±0.09a


A20170701+CJM27+CYB41		 好氧3 d 39.59±0.37b
厌氧3 d 44.26±0.78a
先酶解2 d、再厌氧3 d 36.06±0.40bc


A20170701+CJM27+CMCC		 好氧3 d 26.95±5.04d
厌氧3 d 20.66±0.19e
先酶解2 d、再厌氧3 d 26.26±2.07d


A20170701+CJM27+CYB41+CMCC		 好氧3 d 33.92±0.66c
厌氧3 d 32.69±0.44c
先酶解2 d、再厌氧3 d 45.00±0.20a

A20170701为干酪乳杆菌,CJM27为酵母菌,CYB41为枯草芽孢杆菌,CMCC为黑曲霉。2菌株组合之间以及3菌株和4菌株组合之间分别进行差异显著性比较。

A20170701 was Lactobacillus casei, CJM27 was Saccharomyces, CYB41 was Bacillus subtilis, and CMCC was Aspergillus niger. Significant differences were compared between the 2-strain combinations and between the 3-strain and 4-strain combinations, respectively.

2.4 多菌株接种量正交试验

由表9和表10可知,采用干酪乳杆菌A20170701(A)、酵母菌CJM27(B)、枯草芽孢杆菌CYB41(C)和黑曲霉CMCC(D)4株菌进行混合发酵,对茶皂素脱除率影响的主次因素依次为:B>C>D>A,其中酵母菌CJM27和枯草芽孢杆菌CYB41对茶皂素脱除率具有显著性影响(P<0.05),最优接种组合为A1B1C3D2,即干酪乳杆菌A20170701接种量为1×106 CFU/mL、酵母菌CJM27接种量为1×106 CFU/mL、枯草芽孢杆菌CYB41接种量为1×108 CFU/mL、黑曲霉CMCC接种量为1×107 CFU/mL。
表9 多菌株接种量正交试验结果

Table 9 Results of orthogonal experiment for inoculation volume of multiple strains

项目
Items		 干酪乳杆菌
A20170701
Lactobacillus casei
A20170701 (A)		 酵母菌CJM27
Saccharomyces
CJM27 (B)		 枯草芽孢
杆菌CYB41
Bacillus subtilis
CYB41 (C)		 黑曲霉CMCC
Aspergillus niger
CMCC (D)		 茶皂素脱除率
Removal rate of
tea saponin/%
1 1 1 1 1 53.23±1.98
2 1 2 2 2 54.10±1.05
3 1 3 3 3 51.34±0.18
4 2 1 2 3 54.49±0.64
5 2 2 3 1 53.18±0.73
6 2 3 1 2 48.50±0.06
7 3 1 3 2 56.96±3.73
8 3 2 1 3 49.74±0.35
9 3 3 2 1 49.26±0.55
K1 158.67 164.67 151.47 155.67
K2 156.18 157.02 157.86 159.57
K3 155.97 149.10 161.49 155.58
k1 52.89 54.90 50.49 51.90
k2 52.05 52.35 52.62 53.19
k3 51.99 49.71 53.82 51.87
R 0.90 5.19 3.33 1.32
主次因素Primary and secondary factors B>C>D>A
最优组合Optimal combination A1B1C3D2
表10 多菌株接种量正交试验方差分析

Table 10 Analysis of variance of orthogonal experiment for inoculation volume of multiple strains

项目
Items		 离差平方和
Sum of squares of
deviations		 自由度
Degree of
freedom		 均方
Mean square		 F值
F-value		 P值
P-value
干酪乳杆菌A20170701
Lactobacillus casei A20170701 (A)		 4.534 2 2.267 1.003 0.386
酵母菌CJM27 Saccharomyces CJM27 (B) 121.204 2 60.602 26.810 <0.001
枯草芽孢杆菌CYB41
Bacillus subtilis CYB41 (C)		 51.355 2 25.677 11.360 0.001
黑曲霉CMCC Aspergillus niger CMCC (D) 10.337 2 5.168 2.287 0.130
误差Error 40.687 18 2.260

2.5 酶制剂筛选

采用纤维素酶、木聚糖酶和β-葡聚糖酶,按照每种酶100 U/g的添加量进行单酶、双酶和三酶酶解试验,结果显示(表11),纤维素酶+木聚糖酶+β-葡聚糖酶三酶处理粗纤维降解率优于单酶和双酶处理,其中添加150 U/g纤维素酶、50 U/g木聚糖酶和100 U/g β-葡聚糖酶粗纤维降解率最高,为14.91%。
表11 不同酶制剂对油茶籽粕粗纤维的降解效果

Table 11 Degradation effects of different enzyme preparations on crude fiber in camellia seed meal%

项目
Items		 粗纤维含量
Crude fiber content		 粗纤维降解率
Degradation rate of
crude fiber
未处理油茶籽粕Untreated camellia seed meal 30.63±0.12
纤维素酶Cellulase 26.94±0.29c 12.02±0.93b
木聚糖酶Xylanase 29.93±0.19a 2.28±0.63d
β-葡聚糖酶β-glucanase 28.12±0.10b 8.18±0.33c
纤维素酶+木聚糖酶Cellulase+xylanase 26.80±0.78cd 12.48±2.55ab
纤维素酶+β-葡聚糖酶Cellulase+β-glucanase 28.06±0.05b 8.37±0.15c
木聚糖酶+β-葡聚糖酶Xylanase+β-glucanase 28.12±0.15b 8.18±0.49c
纤维素酶+木聚糖酶+β-葡聚糖酶Cellulase+xylanase+β-glucanase (1∶1∶1) 26.08±0.55d 14.85±1.79a
纤维素酶+木聚糖酶+β-葡聚糖酶Cellulase+xylanase+β-glucanase (1.5∶0.5∶1.0) 26.06±1.26d 14.91±2.74a

2.6 菌酶协同发酵工艺优化

如图2所示,在菌酶协同发酵工艺单因素试验中,随着发酵含水量的增加,茶皂素脱除率先提高后降低,当料水比为1.00∶0.55时,油茶籽粕中茶皂素脱除率最高,为55.08%;随着发酵时间的延长,茶皂素脱除率先提高后降低,在96 h时达到最高,为59.09%;发酵温度为34 ℃时,茶皂素脱除率最高,为59.09%。由表12和表13可知,正交试验结果显示,料水比、发酵时间和发酵温度对茶皂素脱除率均有显著影响(P<0.05),影响的主次因素依次为:料水比(A)>发酵时间(B)>发酵温度(C),最佳发酵工艺组合为A1B1C1,即料水比为1.00∶0.50、发酵时间为84 h、发酵温度为32 ℃。采用所筛选的最佳发酵工艺进行3批次发酵试验验证,茶皂素脱除率分别为61.44%、61.83%和61.64%。
图2 不同料水比、发酵时间和发酵温度对茶皂素脱除率的影响

Fig.2 Effects of different material to water ratios, fermentation time and fermentation temperatures on removal rate of tea saponin

表12 菌酶协同发酵正交试验结果

Table 12 Results of orthogonal experiment for co-fermentation of bacteria and enzymes

项目
Items		 料水比
Material to water
ratio (A)		 发酵时间
Fermentation
time (B)		 发酵温度
Fermentation
temperature (C)		 茶皂素脱除率
Removal rate of
tea saponin/%
1 1 1 1 59.98±0.64
2 1 2 2 55.19±0.05
3 1 3 3 57.21±0.09
4 2 1 2 52.32±0.17
5 2 2 3 46.82±0.43
6 2 3 1 52.57±0.34
7 3 1 3 58.58±0.46
8 3 2 1 56.27±1.21
9 3 3 2 53.84±0.87
K1 172.38 170.88 168.81
K2 151.71 158.28 161.37
K3 168.69 163.62 162.60
k1 57.45 56.97 56.28
k2 50.58 52.77 53.79
k3 56.22 54.54 54.21
R 6.87 4.20 2.49
主次因素Primary and secondary factors A>B>C
最优组合Optimal combination A1B1C1
表13 菌酶协同发酵正交试验方差分析

Table 13 Analysis of variance of orthogonal experiment for co-fermentation of bacteria and enzymes

项目
Items		 离差平方和
Sum of squares of
deviations		 自由度
Degree of
freedom		 均方
Mean square		 F值
F-value		 P值
P-value
料水比Material to water ratio (A) 242.983 2 121.491 93.095 <0.001
发酵时间Fermentation time (B) 79.991 2 39.995 30.647 <0.001
发酵温度Fermentation temperature (C) 31.918 2 15.959 12.229 <0.001
误差Error 26.101 20 1.305

2.7 菌酶协同发酵前后油茶籽粕主要成分变化

由表14可知,油茶籽粕经菌酶协同发酵处理后,主要成分含量发生了显著变化(P<0.05)。与发酵前相比,发酵后油茶籽粕粗蛋白质、粗脂肪和还原糖含量显著提高(P<0.05),分别提高了7.80%、39.77%和168.32%;粗纤维、总糖和总酚含量显著降低(P<0.05),分别降低了13.70%、3.10%和11.64%;同时,总氨基酸含量显著提高(P<0.05),提高了7.97%。
表14 菌酶协同发酵前后油茶籽粕主要成分变化

Table 14 Changes of main components in camellia seed meal before and after co-fermentation of bacteria and enzymes

项目
Items		 发酵前
Before fermentation		 发酵后
After fermentation
粗蛋白质Crude protein/% 7.05±0.04b 7.60±0.06a
粗纤维Crude fiber/% 38.61±0.45a 33.32±0.03b
粗脂肪Crude fat/% 3.52±0.21b 4.92±0.21a
总糖Total sugar/% 19.35±0.03a 18.75±0.02b
还原糖Reducing sugar/(mg/g) 9.69±0.02b 26.00±0.03a
总酚Total polyphenol/(mg/g) 7.73±0.17a 6.83±0.14b
茶皂素Tea saponin/% 15.30±0.54a 5.87±0.57b
总氨基酸Total amino acids/% 7.40±0.04b 7.99±0.08a

3 讨论

3.1 热水浸提油茶籽粕营养价值分析

茶皂素易溶于热水,热水浸提可有效脱除油茶籽粕中的茶皂素。本研究在料水比为1∶11、浸提温度为80 ℃、浸提时间为4 h时茶皂素脱除率最高,为72.75%。在不同浸提温度单因素试验中,当浸提温度高于85 ℃时,茶皂素脱除率反而降低,这与朱俊朋等[19]的研究结果相似,可能是高温造成浸提液的蒸发浓缩。在不同浸提料水比单因素试验中,结果显示在料水比为1∶10之前,随着含水量的增加,浸提效率提高;但是,当达到一定值后,浸提效率不再继续提高,可能是由于随着含水量的增加,营养物质损失更大导致,这与Ma等[12]的结果相似。虽然热水浸提可以有效去除茶皂素,脱除率达到了72.75%,但是浸提后粗蛋白质、总糖和还原糖含量分别降低了20.71%、38.29%和65.63%,营养成分损失较多,而粗纤维含量提高了25.93%,对油茶籽粕营养价值产生较大影响,这与王郝为[20]的研究结果一致。而且,热水浸提茶皂素水分用量较大,处理困难,对环境造成了二次污染。由此可知,热水浸提不是油茶籽粕饲料化应用的最佳处理方法。

3.2 茶皂素脱除菌株筛选及发酵工艺优化

本试验中,筛选得到3株茶皂素降解菌株,分别为酵母菌CJM27、枯草芽孢杆菌CYB41和黑曲霉CMCC,在料水比为1.0∶0.4的情况下发酵3 d,茶皂素脱除率分别达到36.68%、29.49%和31.84%,这与朱晓丽等[13]筛选到的3株菌脱除率相近,其筛选到的3株菌分别为Pleurostoma richardsiae X1-1、蒙氏假单胞菌M4-2(Pseudomonas monteilii M4-2)和多粘类芽孢杆菌M5-2(Paenibacillus polymyxa M5-2),固态发酵油茶籽粕28 d后,茶皂素脱除率分别为34.06%、45.05%和46.15%。任泽文[21]利用黑曲霉L-2进行油茶籽粕固态发酵(含水量80%),发酵3 d后茶皂素脱除率可达80%以上。卫洋洋等[22]利用黑曲霉固态发酵油茶饼粕与豆粕混合物料,在料水比为1.0∶0.5条件下发酵4 d,茶皂素脱除率达到66.3%。管维等[23]采用嗜酸小球菌固体发酵油茶籽粕与麦麸混合物料(含水量70%),发酵5 d后茶皂素脱除率达68.42%。本试验紧密结合生产实际,考虑烘干成本问题,料水比选择1.0∶0.4,可能因为含水量低的原因,各菌株茶皂素脱除率均不太高。
本试验利用2菌株、3菌株和4菌株进行混合发酵发现,3菌株和4菌株混合发酵脱除茶皂素效果优于2菌株。在2菌株发酵中,CYB41+CMCC处理脱除茶皂素效果显著优于其他处理。同时,对3菌株和4菌株混合发酵进行好氧、厌氧以及酶解+厌氧的不同工艺比较发现,先利用纤维素酶酶解2 d,再接种酵母菌CJM27、枯草芽孢杆菌CYB41、黑曲霉CMCC和干酪乳杆菌A20170701厌氧发酵3 d后,茶皂素脱除率最高,达到45.00%。细胞壁是影响茶皂素浸出的主要原因之一,纤维素酶可以破坏油茶籽粕的细胞壁结构,促进茶皂素的释放[24],再利用菌株发酵降解。4菌株组合茶皂素脱除效果优于2菌株和3菌株组合,说明4菌株之间起到了协同促进作用。正交试验得出,4株菌的最佳接种组合为1×106 CFU/mL干酪乳杆菌A20170701、1×106 CFU/mL酵母菌CJM27、1×108 CFU/mL枯草芽孢杆菌CYB41以及1×107 CFU/mL黑曲霉CMCC,其中酵母菌CJM27和枯草芽孢杆菌CYB41对茶皂素脱除率具有显著影响。由此可见,酵母菌CJM27和枯草芽孢杆菌CYB41对茶皂素降解起到了主要作用。

3.3 纤维素降解酶筛选

油茶籽壳层薄而脆,榨油时往往会有部分壳残留,碾粉时可增加摩擦力,起到松散的作用,所以油茶籽粕中粗纤维含量较高,且木质纤维素含量较高。利用纤维素酶、木聚糖酶和β-葡聚糖酶进行单酶、双酶和三酶的固态酶解试验发现,三酶处理对油茶籽粕中粗纤维的降解率最高,达到14.91%。关于油茶籽粕中粗纤维降解的研究较少,王郝为[20]利用600 mg/kg的酸性纤维素酶、400 mg/kg的果胶酶和600 mg/kg的木聚糖酶酶解油茶籽粕,粗纤维降解率可达40.20%。本研究中,粗纤维降解率低于上述研究结果,可能是由于酶添加量较低,或是由于粗纤维结构不同导致。

3.4 菌酶协同工艺优化

本试验经过菌酶协同工艺优化,筛选到最佳菌酶协同发酵工艺为:先加入150 U/g纤维素酶、50 U/g木聚糖酶和100 U/g β-葡聚糖酶,在料水比为1.0∶0.4条件下酶解2 d,然后接种1×106 CFU/mL干酪乳杆菌A20170701、1×106 CFU/mL酵母菌CJM27、1×108 CFU/mL枯草芽孢杆菌CYB41和1×107 CFU/mL黑曲霉CMCC,调节料水比为1.00∶0.50,在32 ℃恒温培养箱中发酵84 h,发酵后茶皂素脱除率达到61.64%。李萌萌[25]采用黑曲霉T5与地衣芽孢杆菌B1混合进行固态油茶籽粕发酵,茶皂素脱除率达到64.88%,与本试验结果相近。孙磊等[26]利用黑曲霉与地衣芽孢杆菌混合发酵,茶皂素脱除率达到54.14%,略低于本试验结果。Fu等[27]筛选到一株柠檬酸杆菌,在液态发酵条件下,茶皂素脱除率达到82.6%。由此可见,液态条件下发酵脱除茶皂素的效果要优于固态发酵。
同时,菌酶协同发酵处理后油茶籽粕粗蛋白质、总氨基酸、粗脂肪和还原糖含量分别提高了7.80%、7.97%、39.77%和168.32%,粗纤维、总糖和总酚含量分别降低了13.70%、3.10%和11.64%,显著提高了油茶籽粕的饲用价值。周浩宇等[28]采用枯草芽孢杆菌、黑曲霉和产朊假丝酵母发酵油茶籽粕8 d,茶皂素脱除率达到87.63%,粗蛋白质含量提高了68.16%,粗纤维含量降低了37.29%,总酚含量降低了87.87%,发酵后营养指标变化高于本试验,可能是由于发酵时间较长导致。

4 结论

综上所述,热水浸提法虽然在脱除茶皂素上具有优势,但是也会显著降低油茶籽粕粗蛋白质、总糖和还原糖含量,提高粗纤维含量,降低了油茶籽粕饲用品质。菌酶协同发酵可提高油茶籽粕粗蛋白质、总氨基酸、粗脂肪和还原糖含量,降低粗纤维含量,提升了油茶籽粕的饲用品质,处理效果优于热水浸提法,并且无废水造成的二次污染。
在本试验条件下,油茶籽粕最佳菌酶协同发酵工艺为:先加入150 U/g纤维素酶、50 U/g木聚糖酶和100 U/g β-葡聚糖酶,在料水比为1.0∶0.4条件下酶解2 d,然后接种1×106 CFU/mL干酪乳杆菌A20170701、1×106 CFU/mL酵母菌CJM27、1×108 CFU/mL枯草芽孢杆菌CYB41和1×107 CFU/mL黑曲霉CMCC,调节料水比为1.00∶0.50,在32 ℃恒温培养箱中发酵84 h,发酵后茶皂素脱除率达到61.64%。

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