一株产木聚糖酶的蜡样芽孢杆菌对雪茄烟叶成分及发酵产物的影响

doi:10.13560/j.cnki.biotech.bull.1985.2021-0349

摘要/Abstract

摘要：

雪茄烟叶中的半纤维素在叶脉及叶片韧性等特性中发挥重要作用,烟叶叶脉较粗,半纤维素含量过高,导致雪茄烟叶韧性较差,可用性变差。本实验室前期筛选到一株产木聚糖酶的蜡样芽孢杆菌,该菌株对烟碱有较好的耐受性,利用液态发酵方法研究菌株以雪茄烟叶为营养源产木聚糖酶的最佳发酵条件,并对最佳发酵条件下菌株降解烟叶中半纤维素的效果进行测定,最后分析比较了菌株对雪茄烟叶香气物质成分及含量的影响。结果表明,菌株在含有不高于1.0 g/L烟碱的培养基中生长受抑制较小,菌株对烟碱有良好的耐受性;当培养基中初始 pH 6.7,接种量为5%,雪茄烟叶底物浓度为20 g/L,发酵24 h,菌株产木聚糖酶活性最高,可达（4.04±0.18）U/mL;在最佳发酵条件下,雪茄烟叶中半纤维素含量为3.38%,降低了6.63%,纤维素含量为10.54%,降低了8.19%,利用菌株发酵同时降低了雪茄烟叶中纤维素和半纤维素含量;通过对香气物质影响的分析比较,发现经菌株发酵后的雪茄烟叶能够提升叶绿醇（2.85%）、油酸（0.62%）、正二十六烷（1.75%）和正三十一烷（14.47%）等物质的含量。蜡样芽孢杆菌 B. cereus 对雪茄烟叶中半纤维素及纤维素降解具有一定作用,后期可应用于雪茄外包皮烟叶的固态发酵,进一步改善雪茄外包皮烟叶的物理特性,为该菌株的开发利用奠定基础。

关键词: 雪茄外包皮烟叶, 木聚糖酶, 蜡样芽孢杆菌, 发酵, 半纤维素

Abstract:

Hemicellulose in cigar wrapper leaves plays an important role in the characteristics of vein and leaf toughness. The thick vein and high hemicellulose content in cigar leaves lead to poor toughness and usability of cigar wrapper. A xylanase-producing strain Bacillus cereus was preliminary screened in the previous stage of our lab,it was found that its tolerance to nicotine was fine. Then the liquid fermentation experiment was used to determine the optimal fermentation condition when the strain was using cigar leaves as nutrient sources to produce xylanase. Under the optimal fermentation condition,the degradation effect of hemicellulose in the tobacco leaves was measured,and finally the effects of the strain on the composition and contents of aroma materials in the cigar leaves were compared and analyzed. The results showed that the growth of the strain was inhibited little in the medium of ≤ 1.0 g/L nicotine,i.e.,the strain had the favorable tolerance to nicotine. When the initial pH of the culture medium was 6.7,the inoculation amount was 5%,and the concentration of cigar leaf substrate was 20 g/L,and fermentation for 24 h,the xylanase production of the strain was the highest,reaching（4.04±0.18）U/mL. Under the optimal fermentation conditions,the hemicellulose content in the cigar leaves was 3.38%,which decreased by 6.63%,and the cellulose content was 10.54%,which decreased by 8.19%, meanwhile,the contents of cellulose and hemicellulose in the cigar leaves decreased under the fermentation. The analysis of aroma materials in the fermented products revealed that phyllol alcohol（2.85%）,oleic acid（0.62%）,n-hexadecane（1.75%）and hentriacontane（14.47%）were enhanced after fermentation by the strain. It is expected that B. cereus can be used in the solid fermentation of leaves for cigar wrapper to improve the physical properties of tobacco leaves,thus laying a foundation for the development and utilization of this strain.

Key words: leaf for cigar wrapper, xylanase, Bacillus cereus, fermentation, hemicellulose

李志豪, 张鸽, 貊志杰, 邓帅军, 李佳轶, 张海波, 刘晓晖, 刘好宝. 一株产木聚糖酶的蜡样芽孢杆菌对雪茄烟叶成分及发酵产物的影响[J]. 生物技术通报, 2022, 38(2): 105-112.

LI Zhi-hao, ZHANG Ge, MO Zhi-jie, DENG Shuai-jun, LI Jia-yi, ZHANG Hai-bo, LIU Xiao-hui, LIU Hao-bao. Effects of a Xylanase-producing Bacillus cereus on the Composition and Fermented Products of Cigar Leaves[J]. Biotechnology Bulletin, 2022, 38(2): 105-112.

图/表 7

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香气物质 Aroma substance	保留时间 Retention time/min	处理 Treatment/%
香气物质 Aroma substance	保留时间 Retention time/min	对照 CK	菌处理 L-1	原始烟叶 The original cigar leaves
1-十八烷基磺酰氯1-Octadecanesulphonyl chloride	11.575	0.1218	0.1296	0.1790
甲氧基乙酸（2-十四酯）Methoxyacetic acid,2-tetradecyl ester	15.04	/	0.1108	/
肉豆蔻醛Tetradecanal	19.309	0.2260	0.2142	0.3164
植酮2-Pentadecanone,6,10,14-trimethyl-	19.563	0.6054	0.6454	0.7955
棕榈酸n-Hexadecanoic acid	20.818	3.0317	1.7673	1.7294
叶绿醇Phytol	22.21	2.3976	2.8542	2.4493
油酸Oleic Acid	22.489	/	0.6200	/
4,8,12,16-四甲基十七烷-4-内酯 4,8,12,16-Tetramethylheptadecan-4-olide	24.324	0.9380	1.3038	1.7624
法尼基丙酮Farnesylacetone	24.514	1.9852	1.6628	2.2967
苯酚,2,2'-亚甲基双-[6-(1,1-二甲基乙基)-4-甲基- Phenol, 2,2'-methylenebis[6-(1,1-dimethylethyl)-4-methyl-	24.853	2.2169	2.5391	3.5442
二十二烷醇Behenic alcohol	25.461	0.9730	1.3907	1.2161
1,6,10-Dodecatrien-3-ol,3,7,11-trimethyl-	28.024	1.5506	1.2632	1.5503
正二十六烷Hexacosane	28.189	1.2867	1.7452	1.2431
正三十四烷Tetratriacontane	28.502	4.4185	5.4284	3.7961
正二十七烷Heptacosane	28.942	0.7448	0.5901	0.7704
正二十三烷Tricosane	29.056	2.0829	2.2008	2.1598
3-二十二烯3-Eicosene,（E）-	29.263	2.0829	1.7491	1.3743
二十五烷Pentacosane	29.897	6.1653	9.5868	6.7700
正三十一烷Hentriacontane	30.316	12.8697	14.4680	11.1620
山嵛醇氯Behenyl chloride	31.06	3.7640	5.1565	/
4,4-二甲基-胆甾烷-6,22,24-三烯 Cholesta-6,22,24-triene, 4,4-dimethyl-	32.122	4.1490	3.1432	3.6484
正二十九烷Nonacosane	32.629	3.9748	3.3805	8.2965
Pyridine-3-carboxamide,oxime,N-（2-trifluoromethylphenyl）-	35.048	0.7591	0.3773	0.4379

香气物质 Aroma substance	保留时间 Retention time/min	处理 Treatment/%
香气物质 Aroma substance	保留时间 Retention time/min	对照 CK	菌处理 L-1	原始烟叶 The original cigar leaves
1-十八烷基磺酰氯1-Octadecanesulphonyl chloride	11.575	0.1218	0.1296	0.1790
甲氧基乙酸（2-十四酯）Methoxyacetic acid,2-tetradecyl ester	15.04	/	0.1108	/
肉豆蔻醛Tetradecanal	19.309	0.2260	0.2142	0.3164
植酮2-Pentadecanone,6,10,14-trimethyl-	19.563	0.6054	0.6454	0.7955
棕榈酸n-Hexadecanoic acid	20.818	3.0317	1.7673	1.7294
叶绿醇Phytol	22.21	2.3976	2.8542	2.4493
油酸Oleic Acid	22.489	/	0.6200	/
4,8,12,16-四甲基十七烷-4-内酯 4,8,12,16-Tetramethylheptadecan-4-olide	24.324	0.9380	1.3038	1.7624
法尼基丙酮Farnesylacetone	24.514	1.9852	1.6628	2.2967
苯酚,2,2'-亚甲基双-[6-(1,1-二甲基乙基)-4-甲基- Phenol, 2,2'-methylenebis[6-(1,1-dimethylethyl)-4-methyl-	24.853	2.2169	2.5391	3.5442
二十二烷醇Behenic alcohol	25.461	0.9730	1.3907	1.2161
1,6,10-Dodecatrien-3-ol,3,7,11-trimethyl-	28.024	1.5506	1.2632	1.5503
正二十六烷Hexacosane	28.189	1.2867	1.7452	1.2431
正三十四烷Tetratriacontane	28.502	4.4185	5.4284	3.7961
正二十七烷Heptacosane	28.942	0.7448	0.5901	0.7704
正二十三烷Tricosane	29.056	2.0829	2.2008	2.1598
3-二十二烯3-Eicosene,（E）-	29.263	2.0829	1.7491	1.3743
二十五烷Pentacosane	29.897	6.1653	9.5868	6.7700
正三十一烷Hentriacontane	30.316	12.8697	14.4680	11.1620
山嵛醇氯Behenyl chloride	31.06	3.7640	5.1565	/
4,4-二甲基-胆甾烷-6,22,24-三烯 Cholesta-6,22,24-triene, 4,4-dimethyl-	32.122	4.1490	3.1432	3.6484
正二十九烷Nonacosane	32.629	3.9748	3.3805	8.2965
Pyridine-3-carboxamide,oxime,N-（2-trifluoromethylphenyl）-	35.048	0.7591	0.3773	0.4379