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).

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