产酸性蛋白酶深红沙雷氏菌的筛选及其在种子萌发中的应用

doi:10.13304/j.nykjdb.2022.0420

摘要/Abstract

摘要：

酸性蛋白酶在工业生产中需求量巨大，但我国产酸性蛋白酶菌株的品种较为单一，因此，亟需开发新型的微生物源蛋白酶和选育高产蛋白酶菌种。从海口红树林土壤中筛选出一株可以在酸性环境下产蛋白酶的菌株，采用福林酚法测定蛋白酶活，经过形态学观察、生理生化实验及16S rRNA序列比对鉴定为深红沙雷氏菌。利用该菌株发酵鱼下脚料制备生物液肥，通过种子萌发实验，研究发酵液的促生效果。结果显示，分离出的产酸性蛋白酶菌株R3可以用于鱼下脚料的发酵，在发酵液稀释5~25倍时，处理60 h均可以促进菜心种子的萌发，与市场购买的液肥效果相同。利用分离出的产酸性蛋白酶菌株发酵鱼下脚料制备生物液肥，实现了对废弃鱼蛋白资源的重新利用，增加了鱼类产品的附加值。

关键词: 酸性蛋白酶, 菌株鉴定, 生物液肥, 种子萌发

Abstract:

Acid protease is of great demand in industrial production. At present， the varieties of acidic protease producing strains in China are relatively rare， so it is urgently needed to develop new microbial protease and select high-yield protease strains. A strain producing protease in acidic environment was screened from mangrove soil in Haikou. The protease activity was determined by folinol method. It was identified as Serratia rubidaea by morphological observation， physiological and biochemical experiment and 16S rRNA sequence blast. The strain was used to ferment fish waste to produce biological liquid fertilizer. The effect of the fermentation liquid as biological liquid fertilizer was evaluated through seed germination promotion experiment. The results showed that the protease producing strain R3 isolated in this study could be used for the fermentation of fish waste. When the fermentation broth was diluted 5 to 25 times， the treatment for 60 h could promote the germination of cabbage seeds， and the germination rate reached the effect of liquid fertilizer. In this study， the isolated strain producing acid protease was used to ferment fish waste to biological liquid fertilizer， which utilized the waste fish protein as the resources and increased the value of fish products.

Key words: acidic protease, strain identification, biological liquid fertilizer, seed germination

中图分类号:

S182

彭欣, 冯璨, 马香, 李宏, 唐燕琼, 李娟娟, 刘柱. 产酸性蛋白酶深红沙雷氏菌的筛选及其在种子萌发中的应用[J]. 中国农业科技导报, 2022, 24(9): 88-95.

Xin PENG, Can FENG, Xiang MA, Hong LI, Yanqiong TANG, Juanjuan LI, Zhu LIU. Screening of Acidic Protease Producing Strains and Its Application in Seed Germination[J]. Journal of Agricultural Science and Technology, 2022, 24(9): 88-95.

图/表 11

表1 不同pH缓冲液组分

Table 1 Components of buffer solution with different pH

pH	Na₂HPO₄/mL	柠檬酸 Citric acid/mL	水Water/mL
3	4.11	15.89	—
4	7.71	12.29	—
5	10.3	9.70	—
6	12.63	7.37	—
7	16.47	3.53	—
8	19.45	0.55	—
9	0.88	5.00^*	14.12
10	3.2	5.00^*	11.8

图1 产酸性蛋白酶菌株的酶活菌圈比注：图中不同小写字母表示在P<0.05水平差异显著。

Fig.1 Enzyme activity and D/d of acid protease producing strainNote： Different lowercase letters in the figure indicate significant difference in enzyme activity at P<0.05 level.

图2 R3菌株形态特征

Fig.2 Morphological characteristics of R3 strain

表2 R3菌株的生理生化特征

Table 2 Physiological and biochemical characteristics of R3 strain

指标Index	特征Characteristics
葡萄糖 Glucose	+
V-P试验 V-P test	+
尿素 urea	-
甲基红试验 Methyl red test	-
吲哚试验 Indole test	-
硫化氢 Hydrogen sulfide	-
蔗糖 Sucrose	+
麦芽糖 Malt dust	+
柠檬酸盐试验 Citrate test	+

图3 R3菌株与其他相近菌株基于16S rRNA基因序列的系统发育树

Fig. 3 Phylogenetic tree among R3 strain and other similar strains based on 16S rRNA gene sequence

图4 不同发酵天数下的产酶活性注：图中不同小写字母表示在P<0.05水平差异显著。

Fig. 4 Enzyme activity at different fermentation daysNote：Different lowercase letters in the figure indicate significant difference at P<0.05 level.

图5 不同pH下的产酶活性注：图中不同小写字母表示在P<0.05水平差异显著。

Fig. 5 Enzymatic activity at different pHNote：Different lowercase letters in the figure indicate significant difference at P<0.05 level.

图6 不同温度下的产酶活性注：图中不同小写字母表示在P<0.05水平差异显著。

Fig. 6 Enzyme activity at different temperatureNote：Different lowercase letters in the figure indicate significant difference at P<0.05 level.

图7 鱼下脚料与水不同比例下的酶活注：图中不同小写字母表示在P<0.05水平差异显著。

Fig. 7 Enzyme activity under different proportion of fish to waterNote：Different lowercase letters in the figure indicate significant difference at P<0.05 level.

表3 种子萌发率 (%)

Table 3 Seed germination rate

处理Treatment		24 h	36 h	48 h	60 h
清水Water		0.00 f	0.00 e	17.14±1.31 d	37.14±1.56 e
发酵稀释液 Fermentation dilution	1×	8.57±0.27 e	8.57±0.34 d	17.14±1.23 d	31.43±1.62 e
	5×	17.14±1.13 d	20±1.09 c	42.86±1.35 c	45.71±1.54 de
	10×	37.14±1.31 b	51.43±2.31 b	71.43±1.32 bc	71.43±2.12 c
	15×	28.57±1.12 bc	48.57±1.73 bc	77.14±2.43 b	80±2.57 b
	20×	25.71±1.24 c	51.43±1.81 b	80±1.94 b	88.57±2.67 a
	25×	0.00 f	0.00 e	20±1.41 d	51.43±2.02 d
氨基酸液肥 Amino acid liquid fertilizer		0.00 f	62.86±2.07 a	88.57±2.41 a	91.43±2.47 a

图8 不同处理组的根长与芽长注：图中不同小写字母表示在P<0.05水平差异显著。

Fig. 8 Root length and bud length of different treatment groupsNote：Different lowercase letters in the figure indicate significant difference at P<0.05 level.

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