CN102864091B - One-bacterium multiple-enzyme bacterial strain as well as screening method and application thereof - Google Patents
One-bacterium multiple-enzyme bacterial strain as well as screening method and application thereof Download PDFInfo
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Abstract
The invention relates to a one-bacterium multiple-enzyme bacterial strain as well as a screening method and an application thereof. The bacterial strain is bacillus subtilis (Bacillus subtilis 1.1111) and is collected in the China center for type culture collection with the collection number of CCTCC (China center for type culture collection) No: M2011286. The bacillus subtilis (Bacillus subtilis 1.1111) can be used for preparing the bacterial strains of nine enzymes, i.e. xylanase, protease, phytase, pectinase, lipase, sweet dew glucanase, glucoamylase and the like, and the yields of the protease, the sweet dew glucanase, amylase and the glucoamylase are very high. Meanwhile, the bacterial strain is proved to have strong endurance capacity on cholate, artificial gastric juice and artificial intestinal juice by simulating the internal cholate environment, the artificial gastric juice, artificial intestinal juice and the animal test, safety and growth simulation capability are shown to a tested animal, and a foundation is laid for effectively improving the enzyme production capability of the bacterial strain, simultaneously generating the xylanase, the protease, the phytase, the pectinase, the lipase, the sweet dew glucanase and the glucoamylase and realizing one-bacterium multiple-enzyme fermentation in the fermentation process. The mutual synergistic effect among various enzymes generated by the bacterial strain is strong, and the bacterial strain can be used as a feed additive to be applied to agricultural production for livestock, fowls, aquatic livestock and the like.
Description
Technical field
The present invention relates to an a kind of bacterium enzyme bacterial strain, also relate to screening method and the application of this bacterial strain simultaneously, belong to microbial technology field.
Background technology
Enzyme is a kind of biological catalyst; fodder enzyme preparation environment-friendly type " green " fodder additives efficient as a class, that have no side effect is widely used in animal produces; application fodder enzyme preparation can improve digestibility and the utilization ratio of feed; improve the production performance of livestock and poultry and aquatic animal; can reduce again the content of nitrogen and phosphorous in movement; protection water body and soil are avoided polluting, and will have very wide application prospect.
Early stage zymin is directly extracted as raw material using animals and plants, but because the animal and plant growth cycle is long, is subject to again the impact of the many factors such as geography, weather and season, is unsuitable for large-scale industrial production.People have turned to sight the main source using microorganism as zymin at present, as the microorganism preparation of amylase and proteolytic enzyme is used in suitability for industrialized production.Early stage compound enzymic preparation is to re-use after composite by single enzyme, and this kind of mode easily causes production cost too high, and quality is unstable, uses inconvenient.
Summary of the invention
The object of the present invention is to provide an a kind of bacterium multienzyme bacterial strain.
Meanwhile, the present invention also aims to provide a kind of screening method of this bacterium multienzyme bacterial strain.
Further, the present invention also aims to provide a kind of application of this bacterium multienzyme bacterial strain.
To achieve these goals, technical scheme of the present invention has adopted an a kind of bacterium multienzyme bacterial strain, this bacterial strain is subtilis (Bacillus subtilis) 1.1111, is preserved in Chinese Typical Representative culture collection center, and its preserving number is CCTCCNO:M 2011286.
Apply classical method for determining bacteria and molecular biology method 16S rDNA it is identified to it is subtilis (Bacillus subtilis) 1.1111, the Chinese Typical Representative culture collection center that is deposited in Wuhan University of Wuhan City of Hubei China province on August 11st, 2011, its preserving number is CCTCC NO:M 2011286.
This subtilis is to showing very strong tolerance to environment such as cholate, simulated gastric fluid, simulated intestinal fluids, laboratory animal safety non-toxic is had no side effect, and can produce zytase, proteolytic enzyme, phytase, polygalacturonase, lipase, sweet dew dextranase, saccharifying enzyme plurality of enzymes, this is conducive to the further exploitation in fodder additives as probiotic bacterium of this bacterial strain, for the mutual relationship between character, each enzyme of the further each enzyme of research, realize a bacterium multienzyme fermentation and have laid a good foundation.
A described bacterium multienzyme bacterial strain can produce zytase, amylase, cellulase, proteolytic enzyme, phytase, polygalacturonase, lipase, sweet dew dextranase, saccharifying enzyme.
Technical scheme of the present invention has also adopted a kind of screening method of a bacterium multienzyme bacterial strain, comprise the following steps: to produce zytase, amylase, cellulase, proteolytic enzyme, phytase, polygalacturonase, lipase, sweet dew dextranase, saccharifying enzyme is screening target, use respectively each enzyme selectivity substratum, have or not transparent circle or hydrolysis circle according to periphery of bacterial colonies, and transparent circle or the diameter (D) of hydrolysis circle and the ratio of colony diameter (d), tentatively determine whether that bacterium producing multi enzyme preparation and high yield enzyme are alive, finally separate from laboratory, in the microorganism resource storehouse that screening is preserved, screen the bacterial strain of a bacterium product multienzyme.
Technical scheme of the present invention also relates to a kind of this bacterium multienzyme bacterial strain in the application of preparing aspect probiotic agent.
Inclined-plane and seed culture medium are in g/L: peptone 10.0, and extractum carnis 5, NaCl 5, inclined-plane adds 15-20, pH7.2 again; Fermention medium is in g/L: Semen Maydis powder 36-44, soybean cake powder 36-44, Na
2hPO
48, (NH
4) 2SO
44, NH
4cl 0.75, CaCl
21.0, pH7.0; Culture condition: 10L fermentation cylinder for fermentation volume is 7L, inoculum size is cumulative volume 10%, temperature is 35-40 ℃, air flow is 3L/L.min, stirring velocity is 200-300r/min, fermentation time 30-50h, in fermented liquid, have a large amount of subtilis viable bacterias, take proteolytic enzyme, sweet dew dextranase, amylase and saccharifying enzyme as main, contain the lytic enzymes such as zytase, amylase, cellulase, phytase, polygalacturonase, lipase, by concentrated fermented liquid dry probiotic bacterium and the prozyme mixed preparation made of 30%-60% starch that add simultaneously.
Meanwhile, technical scheme of the present invention also relates to an a kind of bacterium multienzyme bacterial strain in the application of preparing aspect feed.
The addition of compound enzymic preparation in feed is feed 100-200g per ton.
Described compound enzymic preparation consists of proteolytic enzyme 5000-10000U/g, sweet dew dextranase 1000-3000U/g, saccharifying enzyme 700-3000U/g, zytase 7500-15000U/g, amylase 500-3000U/g, cellulase 200-700U/g, phytase 50-80U/g, polygalacturonase 100-200U/g, lipase 200-500U/g.
Prozyme of the present invention has different mechanism of action, and the interpolation in feed can reduce its anti-oxidant action, improves efficiency of feed utilization, improves animal production efficiency, gives full play to the production performance of animal.The present invention proves that by cholate environment, simulated gastric fluid, simulated intestinal fluid and animal experiment in analogue body this bacterial strain has very strong tolerance to cholate, simulated gastric fluid, simulated intestinal fluid, experimental animal is shown to security and short growing ability, this is the enzymatic productivity that effectively improves this bacterial strain, can produce during the fermentation zytase, proteolytic enzyme, phytase, polygalacturonase, lipase, sweet dew dextranase, saccharifying enzyme etc. simultaneously, realize a bacterium multienzyme fermentation and lay a good foundation.Between the plurality of enzymes that this bacterial strain produces, synergistic action effect is strong mutually, can be used as fodder additives and is applied to the agriculture productions such as poultry, fowl, aquatic animal.
The screening of bacterium producing multi enzyme preparation
1. produce the screening of zytase bacterial strain
The bacterial strain thorn having activated is planted in zytase screening culture medium, at 37 ℃ of constant temperature culture 24h, whether have transparent circle to produce according to periphery of bacterial colonies, judge whether it produces zytase.Measure respectively transparent circle diameter and colony diameter, determine the height of its secretion xylanase activity according to both ratios size, and be more than or equal at 2 o'clock and sentence it as with ratio and produce the high bacterial strain of xylanase activity.
2. the screening of bacteria produced proteinase strain
The bacterial strain thorn having activated is planted on casein agar substratum, in 37 ℃ of incubators, cultivate 24h, whether have casein hydrolysis circle to produce according to periphery of bacterial colonies, judge whether it produces proteolytic enzyme.Measure respectively the diameter of hydrolysis circle and bacterium colony with ruler, determine the height of its secretory protein enzymic activity according to both ratios sizes, and be more than or equal at 2 o'clock with ratio and sentence it as the bacterial strain that protease production is high.
3. the screening of phytase-producing strain
The bacterial strain thorn having activated is planted on phytase primary dcreening operation substratum, in 30 ℃ of incubators, cultivate 48h, whether produce transparent circle according to periphery of bacterial colonies and judge whether it produces phytase.Measure respectively diameter (D) and the colony diameter (d) of transparent circle with ruler, tentatively determine that according to both ratio sizes it produces the height of phytase activity, and be more than or equal to and sentence it as the active high bacterial strain of phytase generating at 2 o'clock with ratio.
4. produce the screening of polygalacturonase bacterial strain
The bacterial strain thorn having activated is planted on polygalacturonase primary dcreening operation substratum, in 37 ℃ of incubators, cultivate 36h, after taking-up, drip several CTAB solution in periphery of bacterial colonies, after standing 10min, observe transparent circle.Whether produce transparent circle according to periphery of bacterial colonies and judge whether it produces polygalacturonase.Measure respectively diameter (D) and the colony diameter (d) of transparent circle with ruler, tentatively determine the height of its generation pectinase activity according to both ratio sizes, and be more than or equal at 2 o'clock with ratio and sentence it as the high bacterial strain of product pectinase activity.
5. the screening of yielding lipase bacterial strain
The bacterial strain thorn having activated is planted on lipase primary dcreening operation substratum, in 37 ℃ of incubators, cultivate 24h, whether produce hydrolysis circle according to periphery of bacterial colonies and judge whether it produces lipase.Measure respectively diameter (D) and the colony diameter (d) of hydrolysis circle, tentatively determine that according to both ratio sizes it produces the height of lipase activity, and be more than or equal to and sentence it as the active high bacterial strain of yielding lipase at 2 o'clock with ratio.
6. produce the screening of mannase bacterial strain
The bacterial strain thorn having activated kind in mannase screening culture medium, is cultivated after 24h for 37 ℃, whether produced transparent circle and size thereof by Congo red aqueous assay periphery of bacterial colonies, as the foundation of producing mannase and screening., and be more than or equal at 2 o'clock and sentence it as with ratio and produce the high bacterial strain of mannosans enzymic activity.
7. produce the screening of saccharifying enzyme bacterial strain
By in bacterial strain thorn kind of the Glycosylase screening culture medium after activation, cultivate 36h for 37 ℃, adopt Gram's iodine solution dyeing, having transparent circle to produce in periphery of bacterial colonies proves that it produces saccharifying enzyme.Measure respectively transparent circle diameter and colony diameter, and be more than or equal at 2 o'clock and sentence it as with ratio and produce the high bacterial strain of diastatic activity.
8. the screening of high yield amylase strain
By on kind of the Zulkovsky starch substratum plate of the bacterial strain thorn after activation, in 37 ℃ of incubators, cultivate 24h, adopt Gram's iodine solution dyeing, whether there is transparent circle to produce according to periphery of bacterial colonies and judge whether it produces amylase.Measure respectively transparent circle diameter and colony diameter, and be more than or equal at 2 o'clock with ratio and sentence it as the diastatic bacterial strain of high yield.9. the screening of High Cellulase Production bacterial strain
9. the screening of High Cellulase Production bacterial strain
By in bacterial strain thorn kind of the cellulase screening culture medium after activation, in 37 ℃ of incubators, cultivate 24h, adopt 1mg/ml congo red staining 1h, then use successively, 1mol/LNaCl solution thoroughly washes away dye liquor 1h, use again 1mol/mlHCl fixation, whether form dyeing circle according to periphery of bacterial colonies, judge whether genus bacillus produces cellulase.Measure respectively dyeing loop diameter and colony diameter, be more than or equal to 2 the cellulase bacterium producing multi enzyme preparation that specific activity is higher that sentences it as with ratio.
One bacterium multienzyme fermentation method can enhance productivity greatly, cost-saving, and because each enzyme derives from same bacterial strain, its security and compound property seem and more have superiority.
The evaluation of bacterium producing multi enzyme preparation
Application plain agar substratum is studied the cultural characteristic of bacterium producing multi enzyme preparation, utilizes gram staining method to study its dyeing property; Utilize the biochemical tests such as sugar fermentating test, IMVIC test, Starch Hydrolysis, gelatine liquefication, hydrogen sulfide, lysine decarboxylase test, arginine dihydrolase test, ornithine decarboxylase test to analyze 3 its physio-biochemical characteristics, and utilize molecular biology method 16S rDNA to identify bacterium producing multi enzyme preparation.
The tolerance of bacterium producing multi enzyme preparation and animal experiment
By cholate environment, simulated gastric fluid, simulated intestinal fluid environment in analogue body, measure respectively the tolerance of bacterium producing multi enzyme preparation in the simulated intestinal fluid environment of the simulated gastric fluid of different cholate content, different pH values, different pH values.And to select healthy mice be laboratory animal, bacterial strain is made to bacteria suspension, and (bacteria suspension viable count is 10
9cFU/mL), establish blank, gavage 5 days continuously, the healthy state of observing small white mouse simultaneously.
Accompanying drawing explanation
Fig. 1 is that bacterium producing multi enzyme preparation 16S rDNA pcr amplification is identified collection of illustrative plates; Swimming lane M is DL2000Marker; Swimming lane 1,2 is bacterium producing multi enzyme preparation 16S rDNA pcr amplification product; Swimming lane 3 negative controls.
The phylogenetic tree of Fig. 2 bacterium producing multi enzyme preparation; ▲ be object bacterial strain.
Embodiment
Below in conjunction with accompanying drawing, the specific embodiment of the present invention is described in further detail
Bacterium producing multi enzyme preparation screening embodiment
1, produce the screening of zytase bacterial strain
The bacterium producing multi enzyme preparation thorn of activation is planted in zytase screening culture medium, in 37 ℃ of incubators, cultivate 24h, whether there is transparent circle to produce according to periphery of bacterial colonies, through observing, find that there is this bacterial strain and produce transparent circle, measure transparent circle diameter (D), colony diameter (d) and the transparent circle diameter of bacterial strain and the ratio (D/d) of colony diameter, test-results is in table 1.
Table 1 zytase decomposer enzyme the selection result alive
2. the screening of bacteria produced proteinase strain
The bacterium producing multi enzyme preparation thorn of activation is planted on casein agar substratum, in 37 ℃ of incubators, cultivate 24h, observe and find that there is this bacterial strain strain generation casein hydrolysis circle.Measure hydrolytic circle (D), colony diameter (d) and the hydrolytic circle of bacterial strain and the ratio (D/d) of colony diameter, test-results is in table 2.
Table 2 proteases for decomposing bacterium enzyme primary dcreening operation alive
3. phytase-producing strain the selection result
The bacterium producing multi enzyme preparation thorn of activation is planted on phytase primary dcreening operation substratum plate, in 30 ℃ of incubators, cultivate after 48h, observation discovery bacterial strain produces transparent circle, and its transparent circle diameter (D), colony diameter (d) and the ratio of the two are in table 3.
Table 3 output value acid enzyme bacterial strain screening result
As shown in Table 3, the D/d value of this bacterial strain is 2.125, can tentatively determine that the activity of these bacterial strain phytase generatings is higher.
4. produce polygalacturonase bacterial strain screening result
The bacterium producing multi enzyme preparation thorn of activation is planted on polygalacturonase primary dcreening operation substratum plate, in 37 ℃ of incubators, cultivate after 36h, observe and find that this bacterial strain produces transparent circle, its transparent circle diameter (D), colony diameter (d) and the ratio of the two are in table 4.
Table 4 produces polygalacturonase bacterial strain screening result
5. yielding lipase bacterial strain screening result
By on the bacterium producing multi enzyme preparation thorn of activation kind of lipase primary dcreening operation substratum, in 37 ℃ of incubators, cultivate after 24h, to observe and find that this bacterial strain produces hydrolysis circle, its transparent circle diameter (D), colony diameter (d) and the ratio of the two are in table 5.
Table 5 yielding lipase bacterial strain screening result
6. produce the screening of mannase bacterial strain
The bacterial strain thorn having activated is planted in mannase screening culture medium, cultivate after 24h for 37 ℃, the Congo red aqueous solution of one deck that falls on flat board finds that periphery of bacterial colonies produces transparent circle, and its transparent circle diameter (D), colony diameter (d) and the ratio of the two are in table 6.
Table 6 produces mannase bacterial strain screening result
7. produce the screening of saccharifying enzyme bacterial strain
By in bacterial strain thorn kind of the Glycosylase screening culture medium after activation, cultivate 36h for 37 ℃, adopt Gram's iodine solution dyeing, there is transparent circle in periphery of bacterial colonies, its transparent circle diameter (D), colony diameter (d) and the ratio of the two are in table 7.
Table 7 produces saccharifying enzyme bacterial strain screening result
8. produce amylase genus bacillus the selection result
By in bacterial strain thorn kind of the Glycosylase screening culture medium after activation, cultivate 36h for 37 ℃, adopt Gram's iodine solution dyeing, there is transparent circle in periphery of bacterial colonies, its transparent circle diameter (D), colony diameter (d) and the ratio of the two are in table 8.
Table 8 produces amylase strain the selection result
9. High Cellulase Production genus bacillus the selection result
Bacterial strain thorn after activation kind is screened in screening culture medium at cellulase, cultivate 24h for 37 ℃, dyed, decolouring and fixation, have transparent circle in periphery of bacterial colonies, and its transparent circle diameter (D), colony diameter (d) and the ratio of the two are in table 9.
Table 9 cellulase decomposer enzyme is lived and is screened
The evaluation embodiment of bacterium producing multi enzyme preparation
1. the observation of bacterium producing multi enzyme preparation cultural characters
Bacterium producing multi enzyme preparation is inoculated on plain agar substratum, cultivate 18～24h at 37 ℃, to its bacterium colony size, edge shape, colony colour, protuberance degree and whether the cultural characters such as dry observe, and utilize gram staining method to study its dyeing property etc.Test-results is in table 10.
Table 10 bacterium producing multi enzyme preparation cultural characters and dyeing property
2. the research of bacterium producing multi enzyme preparation physio-biochemical characteristics
By bacterium producing multi enzyme preparation carry out respectively V.P test, MR test, carbohydrate fermentation test,, catalase test, Starch Hydrolysis test, arginine dihydrolase test, lysine decarboxylase test, ornithine decarboxylase test, Citrate trianion utilization test, hydrogen sulfide production test, indole test, gelatin test etc.Its test-results is in table 11.
Table 11 bacterium producing multi enzyme preparation physiological and biochemical test result
Note :+expression positive reaction ,-expression negative reaction.
3. the preliminary evaluation of bacterium producing multi enzyme preparation
According to the cultural characters of bacterium producing multi enzyme preparation, form, dyeing proterties and physiological and biochemical test result, with reference to " uncle Jie Shi Bacteria Identification handbook " (the 8th edition) and " common bacteria system identification handbook ", it being belonged to kind of an evaluation, can this bacterium producing multi enzyme preparation of preliminary judgement be subtilis.
4. the molecular biology identification of bacterium producing multi enzyme preparation
Extract in bacterium producing multi enzyme preparation and carry out 16SrDNA pcr amplification after total DNA with bacterial genomes DNA rapid extraction test kit, as shown in Figure 1, PCR product carries out sequencing by the raw work in Shanghai to electrophoresis result.From sequencer address, the size of this sequence is 1.501Kb.Sequence in sequence and GenBank is compared, and set up phylogenetic analysis, result as shown in Figure 2.
5. the final evaluation of bacterium producing multi enzyme preparation
According to classical method for determining bacteria, with reference to " the outstanding Bacteria Identification handbook of uncle " and " common bacteria system identification handbook ", in conjunction with the comparison of 16SrDNA identification systems, can determine that this bacterium producing multi enzyme preparation is subtilis.
The tolerance of bacterium producing multi enzyme preparation and animal experiment embodiment
1. the bile tolerance of bacterium producing multi enzyme preparation test
Bacterium producing multi enzyme preparation is seeded in respectively containing 0.1%, 0.2%, 0.3% cholate LB solid medium and cultivates upper cultivation after 4～5 days, observe colony growth situation.Result shows that this bacterial strain still can grow.
2. the resistance to simulated gastric fluid test of bacterium producing multi enzyme preparation
Bacterium producing multi enzyme preparation is made to bacteria suspension, and the inoculum size by 1% is linked into respectively in the simulated gastric fluid of different pH2, pH3, pH4, measures its viable count at different time, and it the results are shown in Table 12.
Table 12 bacterium producing multi enzyme preparation is deposited viable count (mL after gastric juice effect
-1)
Note: the logarithmic value that the data in table are viable count.
3. the resistance to simulated intestinal fluid test of bacterium producing multi enzyme preparation
It is 7.6 simulated intestinal fluid that bacterium producing multi enzyme preparation is linked into respectively to pH value by 1% inoculum size, measures viable count at different time, and it the results are shown in Table 13.
Table 13 bacterium producing multi enzyme preparation is deposited viable count (mL after intestinal juice effect
-1)
Note: the data in table are logarithmic value.
4. the animal experiment of bacterium producing multi enzyme preparation
Bacterium producing multi enzyme preparation is made to bacteria suspension, and continuous irrigation is fed 5 days, and the result of observing afterwards at the 10th day is as table 14.
Table 14 bacterium producing multi enzyme preparation animal test results
Claims (3)
1. a bacterium multienzyme bacterial strain, is characterized in that, this bacterial strain is subtilis (Bacillus subtilis) 1.1111, is preserved in Chinese Typical Representative culture collection center, and its preserving number is CCTCC NO:M2011286.
2. a bacterium multienzyme bacterial strain as claimed in claim 1 is in the application of preparing aspect probiotic agent.
3. a bacterium multienzyme bacterial strain as claimed in claim 1 is in the application of preparing aspect feed.
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| CN103266074B (en) * | 2013-05-27 | 2014-09-17 | 山东蔚蓝生物科技有限公司 | B.subtilis spores strain and application thereof |
| CN103704486B (en) * | 2013-12-27 | 2015-02-25 | 湖南鸿鹰生物科技有限公司 | Poultry enzyme including neutral protease and preparation method thereof |
| CN104328097A (en) * | 2014-07-17 | 2015-02-04 | 河南科技大学 | Method for producing cellulase, mannase and glucoamylase from bacillus subtilis |
| CN104762229B (en) * | 2015-03-24 | 2018-01-09 | 福建省农业科学院农业工程技术研究所 | A kind of bacillus subtilis strain and its application |
| CN105400729A (en) * | 2015-07-16 | 2016-03-16 | 山东省食品发酵工业研究设计院 | Antibacterial bacillus subtilis strain producing xylanase |
| CN106467901A (en) * | 2015-12-01 | 2017-03-01 | 华南理工大学 | A kind of Cellvibrio producing agarase, amylase and xylanase |
| CN105950495B (en) * | 2016-01-31 | 2019-09-20 | 中国热带农业科学院热带生物技术研究所 | One plant of Methylotrophic bacillus and its application in livestock breeding wastewater processing |
| CN105820980B (en) * | 2016-04-25 | 2019-04-05 | 湖北工业大学 | A kind of bacillus of yielding lipase and its application |
| CN107043705A (en) * | 2016-12-30 | 2017-08-15 | 大连医科大学 | Prebiotic bacterial screening method for setting up clinical nutrition microorganism resource storehouse |
| CN109679872A (en) * | 2019-01-09 | 2019-04-26 | 武汉设计工程学院 | The Bacillus subtillis CP-3 separated from cray enteron aisle |
| CN110591943B (en) * | 2019-09-05 | 2021-12-17 | 武汉轻工大学 | Bacillus subtilis capable of producing complex enzyme, composition and application thereof |
| CN112322526B (en) * | 2020-10-30 | 2022-04-08 | 中国热带农业科学院热带生物技术研究所 | Bacillus south China sea with cellulose hydrolysis effect and application thereof |
| CN113088459B (en) * | 2021-04-25 | 2023-03-17 | 天津科技大学 | Heat-resistant high-yield candida tropicalis as well as preparation method and application thereof |
| CN114480566A (en) * | 2022-03-04 | 2022-05-13 | 河南金百合生物科技股份有限公司 | Method for detecting whether bacterial strain produces enzyme |
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| CN101148651B (en) * | 2007-09-06 | 2010-04-21 | 中国科学院微生物研究所 | Clostridium and its culturing method and application |
| CN102100311A (en) * | 2009-12-21 | 2011-06-22 | 沈阳爱特杰牧业有限公司 | Additive for predigestion fermented feed for piglet |
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