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Characterization and Identification of High Quality Lactic Acid Bacteria from Hulless Barley Straw Silage

青稞秸秆青贮饲料中优良乳酸菌的筛选及鉴定



全 文 :!23" !3#
 Vol.23  No.3
! " # $
ACTA AGRESTIA SINICA
   2015$  5%
  May.  2015
犱狅犻:10.11733/j.issn.10070435.2015.03.025
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67

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ˆ

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J‚ôK46Œb’tݽcc6,|8 HG24,LH2_
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犮狅犮犮狌狊狆犲狀狋狅狊犪犮犲狌狊)。J‚qIݽcc6vôf5~30℃,pH3.5~7.0Q3.0%j6.5% NaCl"«28ñ·Â
2

|8HG24,LH2fpHð_3I"«287ôñ·,2,qJ‚Iݽcc6ðâ3ƒy、3½ñj3Õñ/
IHI
。46ݽcc68 HG24jLH2ͽô./、ñ·ûæR,z©ªÞ¤x&âݽcc`。
89:

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&âcâ

ݽc
;<,-=
:S816.53     >?@AB:A     >CD=:10070435(2015)03060709
犆犺犪狉犪犮狋犲狉犻狕犪狋犻狅狀犪狀犱犐犱犲狀狋犻犳犻犮犪狋犻狅狀狅犳犎犻犵犺犙狌犪犾犻狋狔犔犪犮狋犻犮犃犮犻犱
犅犪犮狋犲狉犻犪犳狉狅犿犎狌犾犲狊狊犅犪狉犾犲狔犛狋狉犪狑犛犻犾犪犵犲
CUIZhaoming1,GUOGang1,2,YUANXianjun1,LIJunfeng1,YANGXiaodan1,
DINGLiang1,YUChengqun3,SHAOTao1
(1.InstituteofEnsilingandProcessingofGrass,NanjingAgriculturalUniversity,Nanjing,JiangsuProvince210095,China;
2.ColegeofAnimalScienceandVeterinaryMedicine,ShanxiAgriculturalUniversity,Taigu,ShanxiProvince030801,China;
3.InstituteofGeographicSciencesandNaturalResourcesResearch,ChineseAcademyofSciences,Beijing100101,China)
犃犫狊狋狉犪犮狋:Theobjectivesofthisstudyweretoselectthesuitablelacticacidbacteriaofhighlandbarley
strawsilageandresearchthenewtypeofsilageadditivesinTibetanPlateau.Optimallacticacidbacteria
wereisolatedandscreenedfromhulessbarleystrawsilageinTibetanPlateau.Conventionalmicrobiali
dentificationand16SrRNAsequenceanalysismethodswereusedtoidentifytheselacticacidbacteria.A
totalnumberoffourhomofermentativelacticacidbacteriastrainswereisolated,ofwhichHG24andLH2
wereidentifiedas犔犪犮狋狅犫犪犮犻犾犾狌狊.犘犾犪狀狋犪狉狌犿,LH9as犘犲犱犻狅犮狅犮犮狌狊犪犮犻犱犻犾犪犮狋犻犮犻andLH7as犘犲犱犻狅犮狅犮犮狌狊狆犲狀
狋狅狊犪犮犲狌狊.Alisolatedstrainscouldgrowat5~30℃,pH3.5~7.0and3.0% ~6.5% NaCl.Inaddition,
HG24andLH2gaveapositivereactatpH3.0.ThecharacteristicsoflacticacidbacteriaisolatedinTibet
anPlateauincludedlowtemperature,highacidandhighsaltresistances.Ofaltheselectedstrains,HG24
andLH2weretheeximiousstrainstobeusedassilageadditivewiththeuniqueabilitiesofhighacidpro
ductionandfastgrowth.
犓犲狔狑狅狉犱狊:Hulessbarleystraw;Silage;Lacticacidbacteria
EFGH
:20140714;IJGH:20141203
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(1990),á,ÉW›mô§,úË,mVÏÐc]DOúRÌÍ,Email:cuizhaoming096@163.com;:6x× Author
forcorrespondence,Email:taoshaolan@163.com
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Table1 Microbiologicalanalysisofhulessbarleystraw CFU·g-1FW
&âþâ
Silagesample
„ñ&¼G
Countsofviablemicroorganisms
ݽc
Lacticaacidbacteria
2pc
Aerobicbacteria
kc
Mold
tx
Yeast
&͝/
Hulessbarleystraw
2.1×104 5.6×107 4.2×103 6.9×103
  •(Note):CFU,colonyforminguni;FW,freshweight
2.2 FWcoop,ÝÐ
íîJ‚ØI46ݽcc6IMòHIß+
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ßJ=%æ

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8yúï

+Ž=û

c
šŠMðâE›z
。LH7,LH96þM,<ñ,cš
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Ju

+Ž=û

cšŠMðâE›z

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Table2 Morphologicalcharacteristicsoflacticacidbacteriafromsilage
c6U
Strainnumber
&âŽh
Ensilingtime/d
A2M=
Individualshape
cšHI
Colonycharacteristics
E›z
Transparentzone
HG24 10
H=

ÇA6ßJ=%æ
Shortrod,singleorchain
ÝҖ

6M

8yúï

+Ž=û
Milkywhite,round,centralbulge,smoothsurface
â
Existence
LH2 45
H=

ÇA6ßJ=%æ
Shortrod,singleorchain
ÝҖ

6M

8yúï

+Ž=û
Milkywhite,round,centralbulge,smoothsurface
â
Existence
LH7 45
6þ=


Cocci,opposite
ÝҖ

6M

Ju

+Ž=û
Milkywhite,round,flat,smoothsurface
â
Existence
LH9 45
6þ=


Cocci,opposite
ÝҖ

6M

Ju

+Ž=û
Milkywhite,round,flat,smoothsurface
â
Existence
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NOIñ·FG+›

nâݽcc6ùôÛf
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NaClNOñ·Â2。<+›nâݽcc6ù
ðâ3ƒy

3½j3ÕIHñ

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、αâ2DÆfüá、DnÃü、Dê

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Table3 Characteristicsoflacticacidbacteriafromsilage
HñCharacteristics HG24 LH2 LH7 LH9
M=Shape ݽcRod ݽcRod ݽþcCocci ݽþcCocci
’tabFermentationtype Œb’t Homo Œb’t Homo Œb’t Homo Œb’t Homo
×ò÷ð–Gramstain + + + +
;p\þ£‡Catalase - - - -
xyüÍxGasfromglucose - - - -
yeTemperature
30℃ + + + +
15℃ + + + +
10℃ + + + +
5℃ + + + +
NaCl
6.5% + + + +
3% + + + +
pH
7.0 + + + +
6.0 + + + +
5.0 + + + +
4.0 + + + +
3.5 + + + +
3.0 W W - -
  •:“+”+Ŋñ,“-”+ʼnñ,“W”+ÅƊñ
Note:“+”meanspositive;“-”meansnegative;“W”meansweaklypositive
016
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q4 %&*T;FWco¬ûBÝÐ
Table4 Sugarfermentationprofilesoflacticacidbacteriafromsilage
HñCharacteristics HG24 LH2 LH7 LH9
P=êGlycerol - - - -
j_üêErythritol - - - -
DLëüRibose ++ ++ ++ ++
DlüDXylose - - - -
LlüLXylose - - - -
<‚êAdonitol - - - -
βâ2lüáβMethylxyloside - - - -
xÝüGalactose ++ ++ ++ ++
DxyüDGlucose ++ ++ ++ ++
DaüDFructose ++ ++ ++ ++
DÆfüDMannose ++ ++ ++ ++
Lr‚üLSorbose - - - -
C3üRhamnose - - + +
xÝüêDulcitol - - - -
ðüInositol - - - -
Æfê Mannitol ++ ++ - ++
r‚êSorbitol ++ ++ - ++
αâ2DÆfüáαMethylDmannoside ++ ++ - ++
αâ2DxyüáαMethylDglucoside - - - -
NûÖxyü×NAcetylglucosamine ++ ++ ++ ++
êýÁáAmygdalin ++ ++ ++ ++
naáArbutin ++ ++ ++ ++
þ2áEsculin ++ ++ ++ ++
É~áSalicin ++ ++ ++ ++
M(ÃüCelobiose ++ ++ ++ ++
E‚ü Maltose ++ ++ ++ ++
ÝüLactose ++ ++ ++ ++
"Ãü Melibiose ++ ++ ++ ++
ûüSaccharose ++ ++ ++ ++
ÿüTrehalose ++ ++ ++ ++
püInsulin - - - -
n=ü Melezitose ++ ++ - ++
DVÖüDRaffinose ++ ++ - ++
Ç+Starch - - - -
!üGlycogen - - - -
lüêXylitol - - - -
β²üÃüβGentiobiose + + + -
DnÃüDTuranose ++ ++ - +
D:’üDLyxose - - - ++
DCjüDTagatose - - ++ -
DùÿüDFucose - - - -
LùÿüLFucose - - - -
DLxyü½ÕGluconate W W W W
2Z2xyü½Õ2Cetogluconate - - - -
5Z2xyü½Õ5Cetogluconate - - - -
  •:“++”+Åñ·Â2,“+”+Åñ·,“-”+ŋñ·,“W”+ńÆñ·
Note:“++”meansgrowthwel,“+”meansgrowth,“-”meansnongrowth,“W”meansgrowthweakly
2.4 FWccj16犛狉犚犖犃K‡¨©,c
ݽcc6 HG24,LH2,LH7jLH9íPCR
œùnôKI16SrRNA2üUJuæ·eJ*_
1451bp,1453bp,1462bpj1466bp。16SrRNA
116
! " # $ !23"
u€a+›

+5),HG24jLH2¶ž&Ýc
(犔犪犮狋狅犫犪犮犻犾犾狌狊.犘犾犪狀狋犪狉狌犿 ‘WCFS1’)jðüÝ
c
(犔犪犮狋狅犫犪犮犻犾犾狌狊.狆犲狀狋狅狊狌狊‘1242’)I16SrRNA
2üuæ¬Ùñv}Ø100%;LH7¶ðü.þc
(犘犲犱犻狅犮狅犮犮狌狊.狆犲狀狋狅狊犪犮犲狌狊‘ATCC25745’)I16S
rRNA2üuæ¬Ùñ}Ø100%;LH9¶Ý½.
þc
(犘犲犱犻狅犮狅犮犮狌狊.犪犮犻犱犻犾犪犮狋犻犮犻‘DSM20284’)I
16SrRNA2üuæ¬Ùñ}Ø99.5%。WJ‚Ý
½cc6¶UJJÍݽcc62Þ16SrRNA2
üuæ›Õùú5\(
‰1),€aœÅ HG24j
LH2¶ž&Ýc(犔.狆犾犪狀狋犪狉狌犿)jðüÝc
(犔.狆犲狀狋狅狊狌狊)I5\ñèev_99%;LH7¶ðü
.þc
(犘.狆犲狀狋狅狊犪犮犲狌狊)I5\ñèe_100%;
LH9¶Ý½.þc(犘.犪犮犻犱犻犾犪犮狋犻犮犻)I5\ñèe
_100%。HG24,LH2,LH7,LH9f GenBank8
IρU_KJ779086KJ779089。
<1 FWccj犎犌24,犔犎2,犔犎7犔犎91d8c„0ûÊÆ)‘
Fig.1 PhylogenetictreeshowingtherelativepositionsofstrainsHG24,LH2,LH7,LH9and
relatedspeciesasinferredbytheneighborjoiningmethodwith16SrRNAgenesequences
q5 FWccjo16犛狉犚犖犃K‡¨©,c
Table5 Resultsof16SrRNAgenesequencesoflacticacidbacteria
c6QU
Strainandnumber
2üeî“U
Accessionnumber
è?`
Closestrelatedstrain
¶GenBank8è?`I¬Ùñ
Similaritywiththeclosest
describedspeciesinGenBank/%
HG24 KJ779086 ž&Ýc‘WCFS1’犔 .狆犾犪狀狋犪狉狌犿‘WCFS1’ 100%
ðüÝc
‘1242’犔 .狆犲狀狋狅狊狌狊‘1242’ 100%
LH2 KJ779087 ž&Ýc‘WCFS1’犔 .狆犾犪狀狋犪狉狌犿‘WCFS1’ 100%
ðüÝc
‘1242’犔 .狆犲狀狋狅狊狌狊‘1242’ 100%
LH7 KJ779088 ðü.þc‘ATCC25745’犘 .狆犲狀狋狅狊犪犮犲狌狊‘ATCC25745’ 100%
LH9 KJ779089 ݽ.þc‘DSM20284’犘 .犪犮犻犱犻犾犪犮狋犻犮犻‘DSM20284’ 99.5%
216
!3# ·òó:&͝/&âcâ8oÂݽcIºpQ@ˆ
2.5 ,•oFWccjÒW1Ñ"ÝÐ
J‚ݽcc6Iͽûæô¶ß‰2nÅ,
HG24jLH2ðâ¬ÙIͽûæ,0~8hͽû
æSR
,8~16hœ…—i,16~36h”ÞuT,
LH7jLH9ðâ¬ÙIͽûæ,pHðé))°
i"I”•
。46ݽcc6’t36h¢pH
v}Ø3.7P,f MRSn2"«28 HG24j
LH2ͽô.S2。
<2 FWcoÒW0NOÅ
Fig.2 Acidproductionrateoflacticacidbacteria
  J‚ݽcc6Iñ·ô¶ß‰3nÅ,
HG24jLH2<26ݽcñ·Hû,0~8hËÞ
,8~12hODðßi"†÷I”•,12
h¢5¬Tˆ#,ODð2?ÿ—\,"«Ø36hd
ªØ=[#
。LH7jLH9<26ݽcc6 OD
ðߜ…†÷I”•
,20h¢ODð2?ÿ—\,"
«Ø36hdªØ=[#。46ݽcc6f’t
36h¢IODðv}؉2.4P†。46ݽcc
6f MRSn2"«280~12hIځ!יš
R

+ÍG"Hûù·

5¬Tˆ#0Ø36hODð
2?é)‹—

!›Ý½c}ØTˆ#¢vPTˆ
îfÞpÀ8
。46ݽcc6f’t;S8/â
›œI/I#

!›f MRSn2"«28Iځ
!×tSR

ôvR5¬+ÍIJa#

£+ÍG¥
Rûù·

5æÍñà"ݽ
,pH Rûƒ,vX
O&âûâ8Itxc

kcPQÐÑcIñ·

|8f MRSn2"«28 HG24jLH2ñ·ô
.¬/

<3 FWcoÑ"OÅ
Fig.3 Growthcurveof
lacticacidbacteria
3 œ
ݽcf&â’t;S8ï#~VIx¤

|
`a

G"j!ñÕÖ#&âcâI’t»À

f
_öÙ

ž&+Ž¢ñIݽcG"v‡

<ä+c
²GI0.01%~1%,¥zd}ØÂ2&â’tn
UIzƒG"Éu105CFU·g-1FW[10],H*˜®
x&/a+Ž¢#IݽcG"¬‡

f&â;
S8‹0n_o•c™

ßaݽc‹ômŸ’
t

OcÛÔRûñ·

ŸŠ&âcâ’t»Àã

ƒQ«ïð

?íî8

0]4@&͝/+Ž
¢#ݽcIG"_104CFU·g-1FW,‹ô2Ü
&⠒ t n U V I Ý ½ c G "
(> 105
CFU·g-1FW)。üN,_4&âcâI’t»
À

zWgâ>Iֳۘ*úoÀݽc

£Ý½
cHûn_o•c™

‡¥ÖŽÝ½cO)ônó4’”x¤#“½
Þ(ye
[11],
&âye;ƒ6;Ñ

ù‹£ÞÝ
½’t
。Liu[12],Kim[13],j Weinberg[11]J*<‹
ŒyeWL]
(犛狋狔犾狅狊犪狀狋犺犲狊犵狌犻犪狀犲狀狊犻狊)、[h
(犣犲犪犿犪狔狊)j4EI&â»À5WJK,’,ye
;ƒÝ½c!ñ:š‹/

fƒyNO*úݽ
c<&â’t»ÀI¦Ñ/â>a
[14]。
͸&]
Ñû4@xÿ(ÑÒ

£KäúQ»Ý½cO)
fŠ4@I­¤ý؉@O

ü_Q»Ý½cO)
I©ªñ·ye:š_30℃€,æf&]Ñû4
@

*]Óô†,xyéC10~20℃,çÎÏy
ãà

<Ô<*]&âŽI’tSeQ’t»ÀÍ
ñ~VÕÖ

?íî8Ï&͝/&âcâ8J‚
IݽcôÛf5℃INOñ·,æçf10℃Ž
ñ·lm

2,qÑÒ(8J‚qIݽc<ƒ
yI©­Hñ

_fÑ҃yNO

£¤J‚qI
3ƒyݽc5W&âcâIñͦR‰5’
LH

16SrRNAðâóôj5\†IŒ?ñ,|u
æ5\—Rúæi"

fï2€›†9øéŠ


16SrRNAJrà4©8,¬UâàJIñ&67
v¤:5Wv"Iùú5\Ja
。16SrRNA uæ
JKvPRû

%‡4<„ñ&5WJa@ˆ


16SrRNAÕ1Iùú5\\vP‡ˆ„ñ&f5
\8ISò

¥z:â

f`Jay†

ßa2A
JaÇShI16SrRNAuæŒ?ñàÞ97.5%,
°ø_Ðތ¥A`
[15]。
?íî8
,LH7¶ðü.
þc
(犘.狆犲狀狋狅狊犪犮犲狌狊)Iuæ¬Ùñ_100%,5316
! " # $ !23"
ñèe#_100%,üNLH7Ÿ@ˆ_ðü.þc
(犘.犲狀狋狅狊犪犮犲狌狊),LH9¶Ý½.þc(犘.犪犮犻犱犻犾犪犮
狋犻犮犻)Iuæ¬Ùñ_99.5%,5\ñèe_100%,
üNLH9Ÿ@ˆ_ݽ.þc(犘.犪犮犻犱犻犾犪犮狋犻犮犻)。
HG24jLH2¶ž&Ýc(犔.狆犾犪狀狋犪狉狌犿)jð
üÝc
(犔.狆犲狀狋狅狊狌狊)Iuæ¬Ùñv}Ø
100%,5\ñèev_99%。ž&Ýcjðü
ÝcI16SrRNAuæçâ2bpIã*,+,q
vÑIuæ¬Ùñ

bÿ³‡ˆ`^

€c6M
ò.Qñ\Hñ

ðüÝcvP£¤C3üͽ

ž&Ýc‹£¤C3üͽ

ž&ÝcôÛ£
¤n=ü
,DVÖüjαâ2DÆfüá’tÍ
½

-ðüÝc‹ô
。HG24jLH2ô£¤n=
ü
,DVÖü,αâ2DÆfüájLtͽ

-‹ô£¤C3ü
[16]。
üNWݽcc6
HG24jLH2@ˆ_ž&Ýc(犔犪犮狋狅犫犪犮犻犾犾狌狊狆
犾犪狀狋犪狉狌犿)。?íîJ‚Iݽcc`ºàîfÞ
Ýݽc’tIcâjw»8

f’t;S8ï~
Vx¤

vJ‚Þø`:?
[6,9]。
-Ï&]ÑûH
âI®x&&͝/&âcâ8J‚Iݽcid
ª¸|

¤Þ&âIݽc­ðâ3½ñ/

ñ·ûe
RIHI

vRûƒpH ð,XO‹Â„ñ&G
Ì

ßtx

kc

Ïæ¦Ñ&âcâI’t»À

i­ôºà4£¤)`vøñü

fSàIyeL
MåPQø`NOvvPñ·
[17]。
?ÌÍ+›

J‚ØI46ݽcc6vôf3%j6.5%NaCl
INO
,pHf3.5~7.0ILMåñ·,HG24j
LH2iôfpH_3INOñ·,2,qS/I
3Õj3½ô.

ÝNvª

Ï&]Ñû9ø(
8J‚Iݽcc6ðâ¬<(4#I3ýñ

foÂc6Iºpz{8

ͽjñ·ûæ˜
™š~VIz{

˜Ý½c!ñÂ2I~VHI

‹
ŒIc6îfvàIãR

?íî8ݽcSÝ
½þcðâS/Iͽô.jSRñ·ûe

ͽ
ô.z/I˜ž&ÝcLH2j HG24,ñ·ûe
zRI#˜ž&ÝcLH2j HG24。³2¿
z{
,46ݽcc68ž&Ýcz©!&â
câݽc*ú)c`

ÌÍ+›

ݽc*ú)c`Òpž&Ýc

PçŠc6f&âcâ80ÞJ‚
[17]。
“L­¤
€a+›

‹ŒÝ½cc6f&â8nïIx¤‹
Œ

Ç0£¤¥`ݽcâŽP‹ô}Ø#§2 
I&â>a

ݽc*ú):šù˜ž&Ýcj
|ýݽcc`§«n

¥zFGp¤Ý½þ
c

ߛåØc

.þc

Ýþc

ü_|¬©Þ
&â’tIfÜNO

f’tf#GÌlm

áá!
_’tI¨×ݽc

æ"#&â;S8pH I


Œb’tݽcIG"ãÃùø

ݽcô
’tv·Žh

f’tIïA;S8ïmŸx¤

J‚qI46ݽcc6:?Þ&͝/&â


¬Î0©­Þ&͝/I&â

?íî˜W
5¥zP<46cx_*ú):î{P&͝/_
ûâI&âíî

ÌÍ<46c<&͝/&â’
t»ÀIÕÖ

àJ£¤0]4@&͝/À?

Û
“*]¶®x&/§&âcâIñÍ

‡5e
4)*÷I’“

4 m
  ?íîÒ~¸|‰Ï&]Ñû&͝/&âc
â8J‚Iݽc

J‚qI46ݽc,v_Œ
b’tݽc

ðâ3ƒy

3½ñ/IHI

íä
ú„ñ&@ˆÖ³j16SrRNA uæJK,c6
LH9_ݽ.þ c (犘犲犱犻狅犮狅犮犮狌狊犪犮犻犱犻犾犪犮狋犻犮犻),
LH7 _ðü.þc (犘犲犱犻狅犮狅犮犮狌狊狆犲狀狋狅狊犪犮犲狌狊),
HG24jLH2_ž&Ýc(犔犪犮狋狅犫犪犮犻犾犾狌狊狆犾犪狀
狋犪狉狌犿)。|8ž&Ýcͽô.z/,ñ·ûæ
zR

z©ªÞ¤x&âcâݽc*ú)c`

žŸ>?
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[3] .š,û,´,W––,.*úü"¶Ý½cE]§&â»ÀIÕÖ
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516