以黄浆水为基质的芽孢杆菌制剂制备工艺优化及功效分析

官雪芳; 王琦; 郑琪; 林斌; 黄菊青

doi:10.19303/j.issn.1008-0384.2021.06.008

以黄浆水为基质的芽孢杆菌制剂制备工艺优化及功效分析

doi: 10.19303/j.issn.1008-0384.2021.06.008

官雪芳^{1, 2,},
王琦^{1, 2},
郑琪¹,
林斌^1, ,,
黄菊青^{1, 2}

1.
福建省农业科学院农业工程技术研究所，福建　福州　350002
2.
福建省农产品（食品）加工重点实验室，福建　福州　350002

基金项目: 福建省科技计划公益类专项（2019R1032-4）；福建省财政厅项目（闽财指[2014]680号）；福建省农业科学院科技创新团队建设项目（STIT2017-1-10）

详细信息

作者简介:
官雪芳（1979–），女，助理研究员，主要从事微生物学应用研究（E-mail：guan-619@163.com）

通讯作者:
林斌（1964–），男，研究员，主要从事农业废弃物资源化利用（E-mail：linbin591@126.com）

中图分类号: Q 939.9
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出版历程
- 收稿日期: 2020-11-15
- 修回日期: 2021-04-29
- 网络出版日期: 2021-07-13
- 刊出日期: 2021-06-28

Preparation and Functionalities of Bacillus Agents Cultured on Soymilk Wastewater

GUAN Xuefang^{1, 2
,},
WANG Qi^{1, 2},
ZHENG Qi¹,
LIN Bin^{1
, ,},
HUANG Juqing^{1, 2}

1.
Institute of Agricultural Engineering Technology, Fujian Academy of Agricultural Sciences, Fuzhou, Fujian 　350003, China
2.
Fujian Key Laboratory of Agricultural Produce (Food) Processing, Fuzhou, Fujian 　350003, China

摘要

摘要: 目的利用豆腐黄浆水为发酵原料制备芽孢杆菌制剂，降低芽孢杆菌制剂生产成本，提高菌制剂活菌数。方法利用豆腐黄浆水发酵制备枯草芽孢杆菌SB13和地衣芽孢杆菌BL14菌制剂，并以菌落数为指标，采用单因素试验进行制备工艺优化；通过体外模拟消化及DNS法分析其作为饲料添加剂的功效。结果菌株SB13和BL14在黄浆水基质中均能正常生长，其产孢最佳温度为35 ℃，最适培养时间分别为72 h和48 h，最佳保护剂分别为10%脱脂奶粉和4%蔗糖，最适载体分别为10%麦耚和10%淀粉，最适干燥工艺均为冷冻干燥。此工艺条件下， SB13和BL14菌制剂的活菌数分别为6.40×10⁸和9.80×10⁸ cfu·mL⁻¹，菌存活率分别为97.71%和94.69%。两种菌制剂经过人工胃、胰液处理2 h后，菌存活数在2.55×10⁷～13.10×10⁷ cfu·mL⁻¹，内切葡聚糖酶活保存率均大于39%（胃液）和72%（胰液），并对鸡、猪和牛饲料均有显著的糖化作用。其中，菌株BL14对金黄色葡萄球菌有显著抑制效果。结论实现了黄浆水在芽孢杆菌制剂生产中的创新应用，降低了生产成本；通过工艺优化提高了菌制剂中的单位活菌数；制备的芽孢杆菌制剂有助于促进畜禽对饲料的吸收利用，其中菌制剂BL14还可应用于金黄色葡萄球菌感染的畜禽治疗。
- 芽孢杆菌制剂 /
- 载体 /
- 保护剂 /
- 内切葡聚糖酶 /
- 豆腐黄浆水
Abstract: Objective Fermentation processes utilizing soymilk processing wastewater to prepare feed additives with a high viable bacteria count and desirable functionalities were tested for applications. Method Bacillus subtilis SB13 and B. licheniformis BL14 were cultured in the wastewater. Medium formulation and process optimization were conducted with a single factor experiment determined by the count of viable bacteria in the resulting preparations. Functions as an additive for chicken, pig, and cattle feeds of the preparations was evaluated by an in vitro simulated digestion and a DNS method. Result Both SB13 and BL14 grew normally in the soymilk wastewater. The optimized culture processes for SB13 and BL14 included sporulation temperatures at 35 ℃ for 72 h and 48 h, respectively. To facilitate dehydrating the fermentation broths, a protective agent of 10% skim milk powder was added in the medium for SB13 and 4% sucrose for BL14, as well as a carrier of 10% wheat bran for SB13 and 10% starch for BL14. After completion of the fermentation, the broths were freeze-dried and kept in sealed containers prior to functionality tests. The dried SB13 agent showed a viable bacteria count of 6.40×10⁸ cfu·mL⁻¹ and a survival rate at 97.71%, while BL14 a count of 9.80×10⁸ cfu·mL⁻¹ and a survival rate at 94.69%. Upon the artificial gastric juice and pancreatic juice treatments for 2 h, the two preparations had survived counts ranged from 2.55×10⁷ to 13.10×10⁷ cfu·mL⁻¹. The endoglucanase activity of the preparations was more than 39% retained after the gastric juice treatment and 72% after the pancreatic juice treatment. Both agents displayed significant glycation on feed, and BL14 exhibited a significant inhibitory effect on Staphylococcus aureus. Conclusion Utilizing the wastewater from soymilk processing to culture B. subtilis SB13 and B. licheniformis BL14 resulted in preparations that could become a functional additive to forages for livestock and poultry industry. The process was cost effective and environmentally friendly.
- Bacillus preparation /
- carrier /
- protective agent /
- endoglucanase /
- soymilk processing wastewater

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图 1 菌制剂体外模拟胃液和胰液的存活状况分析

注: 不同小写字母表示 5% 水平差异显著（P＜0.05）。

Figure 1. Bacteria survival of preparations treated in vitro by gastric juice and pancreatic juice

Note：Data with different lowercase letters represent significant difference at 5% level (P＜0.05).

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表 1 不同培养时间的芽孢形成率

Table 1. Bacillus sporulation under varied culture durations （单位：%）

发酵时间 Fermentation time	12 h	24 h	36 h	48 h	72 h	96 h
SB13	—	3.85±1.24 d	25.35±2.42 c	49.36±6.12 b	78.80±3.92 a	76.78±1.56 a
BL14	—	3.34±0.62 d	40.02±2.49 c	70.99±2.45 a	64.73±1.31 b	36.18±1.30 c
注:同行数据后不同小写字母表示差异显著（P＜0.05）。表2～9同。 Note: Data with different lowercase letters on same row represent significant difference at 0.05 level. Same for Tables 2–9.

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表 2 不同培养温度条件下的芽孢形成率

Table 2. Bacillus sporulation under varied culture temperatures （单位：%）

温度 Temperature	20 ℃	25 ℃	30 ℃	35 ℃	40 ℃
SB13	12.97±1.53 d	52.12±1.16 c	75.58±0.37 b	92.48±1.51 a	9.15±1.62 e
BL14	3.00±0.50 e	17.46±1.57 d	62.47±3.31 b	82.01±3.17 a	36.56±2.44 c

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表 3 不同保护剂对菌存活率的影响

Table 3. Survival rates of bacteria cultured in media containing varied protective agents （单位：%）

保护剂 Protective agent	10%甘油 10% glycerin	10%脱脂奶粉 10% skimmed milk powder	10%海藻糖 10% trehalose	10%麦芽糊精 10% maltodextrin	10%蔗糖 10% sucrose	0.5%海藻酸钠 0.5% sodium alginate
BL14	84.43±1.61 b	61.05±3.75 c	85.09±0.96 b	63.18±2.02 c	91.10±2.53 a	64.00±1.65 c
SB13	72.09±3.78 b	94.24±2.07 a	52.60±2.66 d	72.09±5.79 b	62.00±2.66 c	78.59±1.12 b

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表 4 最佳保护剂加入量筛选

Table 4. Optimal addition of protective agent 单位：lg（cfu·mL⁻¹）

加入量 Add amount （m/v）	2%	4%	6%	8%	10%	12%
SB13/脱脂奶粉 Skimmed milk powder	9.15±0.04 d	9.22±0.06 cd	9.28±0.03 bc	9.32±0.06 ab	9.41±0.02 a	9.32±0.01 abc
BL14/蔗糖 Sucrose	9.13±0.07 d	9.73±0.02 a	9.52±0.01 b	9.39±0.04 c	9.33±0.04 c	9.16±0.02 d

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表 5 不同载体对菌存活率的影响

Table 5. Survival rates of bacteria cultured in media containing different carriers （单位：%）

保护剂 Carrier	10%麦麸 10% wheat bran	10%米糠 10% rice bran	10%豆粕 10% soybean meal	10%稻壳 10% rice husk	10%淀粉 10% starch
BL14	83.36±3.57 b	87.35±1.04 ab	66.18±4.07 c	87.48±2.75 ab	92.44±0.87 a
SB13	90.25±1.07 a	43.49±2.05 c	85.28±2.36 a	89.56±3.04 a	63.56±2.19 b

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表 6 最佳载体加入量筛选

Table 6. Optimal addition of carrier 单位：lg（cfu·mL⁻¹）

加入量 Add amount（m/v）	2%	4%	6%	8%	10%
SB13/麦麸 Wheat bran	9.35±0.01 d	9.45±0.05 c	9.51±0.11 b	9.54±0.02 b	9.60±0.02 a
BL14/淀粉 starch	9.45±0.02 c	9.49±0.04 bc	9.52±0.03 b	9.56±0.03 ab	9.61±0.00 a

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表 7 不同干燥条件对菌存活率的影响

Table 7. Bacteria survival rates under varied drying conditions （单位：%）

干燥工艺 Drying process	常温烘干 Room temperature drying	喷雾干燥 Spray drying	冷冻干燥 Freeze drying	60 ℃热泵干燥 60 ℃ heat pump drying
SB13	93.59±0.12 a	65.72±3.56 c	95.00±0.49 a	77.76±5.39 b
BL14	91.01±1.69 a	63.56±1.59 c	94.52±5.18 a	74.59±0.95 b

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表 8 几种保护剂在冷冻干燥条件下SB13存活情况

Table 8. Bacteria survival rates of freeze-dried SB13 preparation with various protective agents

项目 Item	原液 Stock solution	10%脱脂奶粉 10% skimmed milk powder	10%麦麸 10% wheat bran	10%脱脂奶粉+10%麦麸 10% skimmed milk powder + 10% wheat bran
SB13存活率 Survival rate of strain SB13/%	37.50±1.36 b	92.59±2.62 a	91.13±3.42 a	97.71±4.32 a
菌落数 Number of colonies/（×10⁸ cfu·mL⁻¹）	1.95±0.07 c	5.00±0.14 b	5.65±0.21 ab	6.41±0.28 a

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表 9 几种保护剂在冷冻干燥条件下菌BL14存活情况

Table 9. Bacteria survival rates of freeze-dried BL14 preparation with various protective agents

项目 Item	原液 Stock solution	4%蔗糖 4% sucrose	10%淀粉 10% starch	4%蔗糖+10%淀粉 4% sucrose + 10% starch
菌BL14存活率 Survival rate of strain BL14/%	36.11±1.96 b	90.20±4.16 a	92.75±4.1 a	94.69±1.37 a
菌落数 Number of colonies/（×10⁸ cfu·mL⁻¹）	2.59±0.14 c	9.24±0.42 b	9.58±0.43 a	9.89±0.14 a

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表 10 酶菌制剂处理对饲料总糖含量的影响

Table 10. Effect of preparation addition on total sugar content of feed （单位：mg·mL⁻¹）

饲料种类　　 Feed species	发酵时间 Fermentation time/h	SB13		BL14
饲料种类　　 Feed species	发酵时间 Fermentation time/h	基础培养基 Basic medium	黄浆水培养基 Soybean wastewater medium	基础培养基 Basic medium	黄浆水培养基 Soybean wastewater medium
猪饲料 Pig feed	0	1.09±0.02 d	1.12±0.08 cd	1.10±0.07 c	1.09±0.05 c
	2	1.21±0.02 b	1.27±0.05 a	1.21±0.02 ab	1.19±0.01 ab
	9	1.15±0.03 bcd	1.19±0.07 bc	1.19±0.03 b	1.15±0.03 bc
	12	1.13±0.01 cd	1.10±0.02 d	1.26±0.09 a	1.10±0.00 c
	24	1.16±0.02 bcd	1.16±0.01 bcd	1.15±0.01 bc	1.16±0.04 bc
鸡饲料 Chicken feed	0	0.79±0.01 d	0.79±0.03 d	0.81±0.00 cd	0.81±0.04 cd
	2	1.29±0.03 a	1.26±0.00 abc	1.27±0.01 b	1.26±0.00 b
	9	1.28±0.00 ab	1.31±0.00 a	1.34±0.00 a	1.30±0.00 ab
	12	1.22±0.00 c	1.23±0.04 bc	1.27±0.00 b	1.29±0.06 ab
	24	0.76±0.03 d	0.79±0.04 d	0.85±0.02 c	0.78±0.03 d
牛饲料 Cattle feed	0	0.77±0.02 f	0.78±0.01 ef	0.78±0.00 e	0.77±0.02 e
	2	0.82±0.01 e	0.91±0.01 d	0.85±0.00 d	0.87±0.03 d
	9	0.97±0.03 c	0.90±0.01 d	0.99±0.05 bc	1.00±0.05 bc
	12	1.18±0.05 b	1.18±0.03 b	0.98±0.00 bc	0.96±0.03 c
	24	1.16±0.04 b	1.26±0.02 a	1.11±0.02 a	1.03±0.05 b
注：不同字母表示不同发酵时间和不同培养基之间差异显著（P＜0.05）。 Note：Different lowercase represent significant difference at 5% level between different fermentation times and different media （P＜0.05）.

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表 11 菌BL14对病原菌的抑制作用

Table 11. Inhibitory effect of BL14 on pathogens

病原菌 Pathogen	抑菌圈直径 Zone of inhibition	病原菌 Pathogen	抑菌圈直径 Zone of inhibition
金黄色葡萄球菌 Staphylococcus aureus	14 mm	大肠杆菌 Escherichia coli	—
单核细胞增生李斯特菌 Listeria monocytogenes	—	宋内氏志贺细菌 Shiga sonnei	—
奇异变形杆菌 Proteus mirabilis	—	鼠伤寒沙门氏菌 Salmonella typhimurium	—
注：“—”表示没有抑菌效果。 Note: "—" means no antibacterial effect.

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