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Development of technique to circularly produce enzyme-probiotics-oligosaccharide mixture, and solutions to replace antibiotics in a low-cost way
Dr. Nicole ZhouSUNHY BIOLOGY CO., LTD, CHINA
EnzymeY - Increase the digestibility of feedN - Treat disease
ProbioticsY - Bacterial resisting functionN - Increase the bioburden
OligosaccharideY - Food for probioticsN - Expensive and late-responsive
Alternative to Antibiotics, solution?
Cost?
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about
about
about
NO MORE Colistin Sulfate IN FEED from 2017 in CHINA!
Enzymes for Feed Enzymes for Oligosaccharides
Screening Strains
By High Throughput ScreeningBy Shotgun Sequencing and Genome-walking Method
Enzymes Products
By High Density Fermentation
Oligosaccharides Fermentation residue
Probiotics
Enzyme-Probiotics-Oligosaccharides
Cost - Effective Solutions
Technical route:
Results:
1. We obtained that such strains can be used to produce xylo-oligosaccharide,
mannan-oligosaccharide and oligogalacturonic acid, respectively. In addition,
mannanase and xylanase can also be added to the feed directly to increase the
digestibility of diet;
By High Throughput ScreeningBy Shotgun Sequencing and Genome-walking Method
Acid xylanaseregistered in EFSA
Mananase screened by Droplet Microfluidics
Alkaline xylanasefor XOS
Alkaline pectinasefor OGA
( GMO ) ( NGMO ) ( NGMO ) ( NGMO )
( R&D Center of Sunhy )
Results:
2. By fermentation process optimization, the activity of xylanase was increased from
7,882 u/mL to 37,129 u/mL, the activity of pectinase was increased from 1,526 u/mL
to 2,190 u/mL, the activity of mannanase was increased from 6,435 u/mL to 10,235
u/mL, which led to the decrease in the cost for enzyme production by 34.77~97.93%;
DO
( ) ( )CCakppaHkN −=−= ** LLA
Flow field analysis
β-Mananase
发酵罐批号GLJTM1407-308-6P
日期 时间 pH C(g/l) P(%) N(%) 检验 备注
703 19:00 4.92 1.04 0.49 李菱澌
日期 时间 周期(h) pH OD600 湿重(%) 酶活(U/mL)
704 07:00 12 4.80 3.57 邝永洁
14:00 19 4.90 35.50 10.72 周翔 甘前
17:00 22 4.91 12.17 周翔
705 07:00 36 4.75 26.56 邝永洁
09:00 39 4.86 145.00 27.25 周翔 甲前
17:00 46 5.05 28.78 周翔
706 07:00 60 5.02 27.83 周翔
17:00 70 4.98 26.72 邱韵心
707 07:00 84 4.97 26.11 2298 邱韵心
17:00 94 5.02 26.02 3064 周翔
708 07:00 108 5.04 26.07 3807 周翔
16:00 117 4.99 26.11 4451 邝永洁
709 07:00 132 5.05 25.66 5089 周翔
16:00 141 4.88 25.70 5396 邝永洁
710 07:00 156 4.90 25.78 6416 邝永洁
16:00 165 4.80 25.84 7029 周翔
711 07:00 180 4.95 26.64 7697 刘荣
16:00 189 4.96 26.19 8653 邝永洁
712 07:00 204 4.89 25.44 9087 张潇
16:00 213 4.80 25.78 10235 邝永洁
含固
6435u/mL 9532u/mL
10235u/mL
30L
60T发酵罐批号
GJM1403-201-7P
日期 时间 pH C(g/l) P(%) N(%) 检验 备注
330 4.65 0.62 0.4 陈志荣
日期 时间 周期(h) pH OD600 湿重(%) 酶活(U/mL)
330 17:00 2 5.68 陈志荣
331 04:15 14 5.72 49.70 11.82 陈志荣 甘前
07:00 17 5.74 66.10 12.55 陈志荣
17:00 27 5.77 97.60 21.98 张荣
401 01:30 36 5.75 158.20 26.56 刘荣 甲前
07:00 41 5.85 27.47 刘荣
17:00 51 5.55 188.75 26.45 陈志荣
402 07:00 65 5.83 200.00 26.54 陈志荣
17:00 75 5.87 230.00 28.61 周翔
403 07:00 89 5.99 260.00 29.33 590 陈志荣
17:00 99 5.96 265.00 29.43 662 周翔
404 07:00 113 6.00 267.00 30.22 816 周翔
17:00 123 5.92 270.00 29.94 920 李菱澌
405 07:00 137 5.98 272.50 30.67 1292 李菱澌
17:00 147 5.99 270.00 30.72 1388 周翔
406 07:00 161 6.04 277.50 31.36 1434 周翔
17:00 171 6.03 280.00 32.66 1472 陈志荣
407 07:00 185 6.08 300.00 32.09 1592 周翔
17:00 195 6.04 282.50 32.14 1720 陈志荣
408 07:00 209 6.05 252.50 31.40 1880 陈志荣
17:00 219 6.05 235.00 32.08 1992 邱韵心
409 09:00 235 6.05 290.00 32.46 2108 陈志荣 放
含固 12.68%
1526u/mL 2190u/mL
2108u/mL
Pectinase
30L
60T
30L
60T
7882u/mL 22580u/mL
37129u/mL
Xylanase
发酵罐批号MJTM1407-201-13P
日期 时间 pH C(g/l) P(%) N(%) 检验 备注
708 02:50 4.53 2.11 0.33 周翔
日期 时间 周期(h) pH OD600 湿重(%) 酶活(U/mL)
708 07:00 4 4.94 2.67 周翔
16:00 13 4.84 7.12 邝永洁
20:00 17 4.99 51.40 5.68 周翔 甘前
709 07:00 28 5.00 20.79 周翔
16:00 37 4.95 159.00 27.57 邝永洁 甲前
710 07:00 52 4.95 26.60 邝永洁
16:00 61 4.81 26.23 周翔
711 07:00 76 4.98 27.40 10866 刘荣
16:00 85 4.89 27.85 13037 邝永洁
712 07:00 100 4.90 27.22 16184 张潇
16:00 109 4.89 28.06 20511 邝永洁
713 07:00 124 4.90 28.17 24138 张渊
16:00 133 4.89 28.16 27219 张潇
714 07:00 148 4.90 28.79 30366 陈志荣
16:00 157 4.91 27.88 32119 邝永洁
715 07:00 172 4.92 28.09 34189 邝永洁
16:00 181 4.98 27.66 35970 周翔
716 07:00 196 4.95 27.28 37129 周翔
16:00 205 4.86 26.53 37419 邝永洁
717 07:00 220 4.95 26.38 36053 周翔
16:00 229 4.91 26.23 35639 邝永洁
含固
Lower-cost for enzymes!
( R&D Center of Sunhy, 2015 )
Corn Cob Meal XOS Solution XOS ( Yield:20% )Alkaline xylanase Spray drying
Pectin
Mananase
Filtratedby
CeramicMembrane
Spray drying
Konjaku flour
MOS( Yield : 95% )
OGA ( Yield : 90% )
Analysis of products
( R&D Center of Sunhy, 2016 )
The shadow cost of enzymes in these process is almost zero.
Molecular weights are between 301.0 to 621.0, that means the substances detected are oligosaccharids.
Results:
3. Using the residues from oligosaccharide production as the medium for fermentation:
Bacillus licheniformis (ACCC 06149) : from 14.2 to 17.8 billion;
Bacillus subtilis (ACCC 11088) : from 3 to 13.7 billion
Clostridium butyricum (ACCC 10390) : from 0.253 to 0.446 billion.
Bacillus licheniformis(ACCC 06149)
Clostridium butyricum(ACCC 10390)
Bacillus subtilis(ACCC 06149)
( Joint R&D Center of Sunhy and Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, 2015 )
Cheaper medium;Higher productivity;Lower cost.
Inhibition of E. coli by Colistin Sulfate(100ppm);
Inhibition of E. coli by Clostridium butyricumE.coli-E80
SalmonellosisC5
Pasteurella gallinarum-C51
Inhibition of Salmonellosis by Colistin Sulfate (100ppm) and Olaquindox (150ppm);
Inhibition of Salmonellosis by Clostridium butyricum
Inhibition of Pasteurella gallinarum by Colistin Sulfate (100ppm) ,Virginiamycin(25ppm), Flavomycin (8ppm) and Zinc bacitracin (40ppm);
Inhibition of Pasteurella gallinarum by Clostridium butyricum
( R&D Center of Sunhy, 2016 ) Colistin Sulfate (100ppm), Olaquindox (150ppm), Virginiamycin (25ppm),
Flavomycin (8ppm) , Zinc bacitracin (40ppm) and Chlorotetracycline
Bacillus licheniformisand
Bacillus subtilis?
Results:
4. By supplementing the enzyme-probiotics-oligosaccharide mixture in weaningDuroc×Landrace×Yorkshire piglets diets (120 piglets, started at 21±2 day-old), we gained the following benefits:
4.1 Reduction of the use of Colistin Sulfate in weaning piglet feed by 20-40 ppm;
4.2 Decrease in FCR by 0.18 (p < 0.05);
4.3 Decrease in diarrhea rate from 2.97% to 1.90% (p < 0.05);
4.4 Increase in ADG by 23 g (p < 0.05).
( R&D Center of Sunhy, 2015 )
GroupsControl group Group 1 Group 2
( 100ppm CS) ( Mixture+80ppm CS) ( Mixture+60ppm CS)
Initial weight, kg 6.40±0.39 6.42±0.14 6.39±0.43
Final weight, kg 11.23±0.99 11.91±1.05 11.72±1.22
ADG, g 230.05±14.59a 261.43±16.01b 253.81±17.61b
ADFI, g 326.60±19.22 316.22±19.74 314.72±19.72
F/G 1.42±0.08a 1.21±0.08b 1.24±0.07b
Diarrhea rate, % 2.97±0.12a 1.14±0.05b 1.90±0.09b
√ Commercial Choice
Why not decrease Colistin Sulfate more?
Commercial Application:
Control
Test
( New Hope Group,2015; Yangxiang Group,2014; Haid Group, 2015)
Discussion:
1. Alkaline xylanase may be not be so good for XOS production, we need to try other xylanase or raw materials;
2. Since only Clostridium butyricum can kill the material, why do we supplement Bacillus licheniformis and Bacillus subtilis together? Because we need to use their metabolites: some digestive enzymes. So, why not supplement digestive enzymes directly;
3. According to our recent results, stabilized lactobacillus is also beneficial to replace antibiotics, such as Enterococcus faecalis;
Discussion:
4. The replacement of 20-40ppm Colistin Sulfate in weaning piglet is not enough, we need to try more.
5. Since we supplemented enzymes, probiotics and oligosaccharides in the feed, we should test the influence of the microbes in the GI, we have done in layer (Chart 1), but not yet in piglet.
Chart 1: high-throughput sequencing of microbes in layer
( R&D Center of Sunhy, 2015 )
Conclusion:
1. Circum-production of the mixture of
enzyme, probiotics and oligosaccharide
is an alternative and low-cost solution to
replace antibiotics
2. It can reduce the enzymes cost of
oligosaccharides production;
3. It also can reduce the cost of probiotics
by higher productivity and cheaper
fermentation medium ;
4. it can reduce the use of Colistin Sulfate in weaning piglet feed by 20-40 ppm,
decrease the FCR by 0.18 (p < 0.05), decrease the diarrhea rate from 2.97% to
1.90% (p < 0.05) and increase the ADG by 23 g (p < 0.05).