Cloning and sequencingof rDNA ampli

Cloning and sequencing
of rDNA amplicons revealed that meso-
D. Ercolini / Journal of Microbiological Methods 56 (2004) 297–314 303
philic bacteria, including leuconostocs, L. lactis, and
Macrococcus caseolyticus were dominant in raw milk,
while S. thermophilus prevailed during the fermentation.
Other rod-shaped LAB, especially L. fermentum
and L. delbrueckii, were also found during ripening.
The technique has also been profitably used by
Cocolin et al. (2001c) to monitor the dynamic changes
in bacterial community during the ripening of natural
fermented Italian sausages by using DNA and RNA
directly extracted from the food matrix. The region V1
of the 16S rDNA was taken into account for the
analysis of the meat samples, and PCR and RT-PCR
were used to obtain amplicons to be separated by
DGGE. Lactic acid bacteria were found in the early
stage of the ripening along with other organisms,
mainly members of the family Micrococcaceae and
meat contaminants, such as Brochothrix thermosphacta
and Enterococcus spp. LAB represented the
main population, especially Lactobacillus sake and L.
curvatus, and were responsible for the acidification
and proteolysis that determined the organoleptic features
of the fermented sausages, while Micrococcaceae
were shown to have a restricted importance
compared to LAB.
The microbial community occurring during vanilla
curing in Indonesia was also studied by this approach
(Ro¨ling et al., 2001). The authors found a significant
occurrence of Bacillus isolates and also highlighted
the presence of unculturable microorganisms during
the high-temperature phases of the bean curing.
Drinks may also benefit from this type of investigation
for both fermented and nonfermented beverages.
LAB communities existing during the
fermentation of Malt whisky were monitored by
van Beek and Priest (2002) by PCR-DGGE of V3
and RT-PCR-DGGE of V6–V8 regions of 16 rDNA
coupled with traditional isolation procedure, viable
staining counts, and scanning electron microscopy.
The authors showed that mainly lactobacilli played
an important role in the fermentation and that
homofermentative Lactobacillus acidophilus and L.
crispatus dominated the last phase of the fermentation.
van Beek and Priest (2002) also optimized the
separation of lactobacilli in DGGE by adopting the
V6–V8 region of 16S rDNA as a target, giving
better resolution of several species due to higher
heterogeneity in sequence among species of the
genus Lactobacillus.
Among microbial food processes, dough leavening
by using sourdoughs as starter is often employed,
yielding appreciated bakery products. Recently, sourdough
fermentation performed by different starters
was monitored by using Lactobacillus group-specific
16S rDNA primers in PCR followed by DGGE
analysis (Meroth et al., 2003). Shifts of Lactobacillus
population during the fermentation of each dough
could be determined and the dominant species carrying
out the process up to the end were identified as
Lactobacillus sanfranciscensis, Lactobacillus pontis,
Lactobacillus mindensis, L. crispatus, Lactobacillus
johnsonii, Lactobacillus frumenti, and Lactobacillus
reuteri, depending on the type of starter used.
6. Identification of members of the cultivable
community: an alternative to isolation
The PCR-DGGE can be also used to rapidly check
the diversity of the bacterial community after cultivation
in liquid or solid and specific or nonspecific
culture media. Briefly, after colony counting has been
performed, the colonies from the plates can be collected
in ‘‘bulks’’ and subjected to DNA extraction
and PCR-DGGE analysis (Ercolini et al., 2001b).
Consequently, a DGGE fingerprint can be obtained
for each plate, each dilution, and culture medium.
This method to investigate the cultivable microbial
community has been shown to have good potential in
food microbiology. Firstly, it provides an alternative
to traditional tools for the identification of dominant
species. Qualification of the dominant species could
be achieved by sequencing the DGGE bands arising
from the patterns corresponding to the highest dilutions
in spite of the isolation of single colonies
followed by purification and biochemical identification.
The bulk from countable plates can be thus used
to identify dominant microbial species from the food
matrix as performed by Ercolini et al. (2003a) in
analysing the Stilton cheese. The advantage is that,
while sequencing of partial 16S rDNA fragments
represents a sound tool for identifying species, identification
of species using phenotypic methods such as
sugar fermentation patterns may sometimes be uncertain,
complicated, and time-consuming, particularly
within LAB, due to an increasing number of species
that vary in a few traits (Quere et al., 1997).

Kết quả (Việt) 1:

Nhân bản và xác định trình tựcủa rDNA amplicons tiết lộ rằng meso-Mất Ercolini / tạp chí vi sinh phương pháp 56 (2004) 297-314 303Anh vi khuẩn, bao gồm cả leuconostocs, L. lactis, vàMacrococcus caseolyticus đã thống trị trong sữa nguyên,trong khi S. thermophilus chiếm ưu thế trong sự lên men.Khác hình que LAB, đặc biệt là L. fermentumvà L. delbrueckii, cũng được tìm thấy trong chín.Các kỹ thuật cũng đã được lợi sử dụng bởiCocolin et al. (năm 2001 của c) để theo dõi những thay đổi năng độngtrong các cộng đồng vi khuẩn trong chín tự nhiênlên men ý xúc xích bằng cách sử dụng DNA và RNAchiết xuất trực tiếp từ ma trận thực phẩm. Vùng V1những 16 rDNA được đưa vào tài khoản cho cácphân tích của thịt mẫu, và Đảng Cộng sản Romania và Đảng Cộng sản Romania RTđược sử dụng để có được amplicons được tách ra bởiDGGE. Axit lactic vi khuẩn được tìm thấy vào đầu những nămgiai đoạn của chín cùng với các sinh vật khác,chủ yếu là thành viên của gia đình Micrococcaceae vàchất gây ô nhiễm thịt, chẳng hạn như Brochothrix thermosphactavà Enterococcus spp. phòng thí nghiệm đại diện cho cácdân số chính, đặc biệt là vì lợi ích Lactobacillus và L.curvatus, và đã được chịu trách nhiệm về quá trình axit hóavà proteolysis xác định các tính năng sốxúc xích lên men, trong khi Micrococcaceaeđược hiển thị để có tầm quan trọng bị giới hạnso với phòng thí nghiệm.Cộng đồng vi sinh vật xảy ra trong vanichữa ở Indonesia cũng được nghiên cứu bởi cách tiếp cận này(Ro¨ling et al., 2001). Các tác giả tìm thấy đáng kểoccurrence of Bacillus isolates and also highlightedthe presence of unculturable microorganisms duringthe high-temperature phases of the bean curing.Drinks may also benefit from this type of investigationfor both fermented and nonfermented beverages.LAB communities existing during thefermentation of Malt whisky were monitored byvan Beek and Priest (2002) by PCR-DGGE of V3and RT-PCR-DGGE of V6–V8 regions of 16 rDNAcoupled with traditional isolation procedure, viablestaining counts, and scanning electron microscopy.The authors showed that mainly lactobacilli playedan important role in the fermentation and thathomofermentative Lactobacillus acidophilus and L.crispatus dominated the last phase of the fermentation.van Beek and Priest (2002) also optimized theseparation of lactobacilli in DGGE by adopting theV6–V8 region of 16S rDNA as a target, givingbetter resolution of several species due to higherheterogeneity in sequence among species of thegenus Lactobacillus.Among microbial food processes, dough leaveningby using sourdoughs as starter is often employed,yielding appreciated bakery products. Recently, sourdoughfermentation performed by different starterswas monitored by using Lactobacillus group-specific16S rDNA primers in PCR followed by DGGEanalysis (Meroth et al., 2003). Shifts of Lactobacilluspopulation during the fermentation of each doughcould be determined and the dominant species carryingout the process up to the end were identified asLactobacillus sanfranciscensis, Lactobacillus pontis,Lactobacillus mindensis, L. crispatus, Lactobacillusjohnsonii, Lactobacillus frumenti, and Lactobacillusreuteri, depending on the type of starter used.6. Identification of members of the cultivablecommunity: an alternative to isolationThe PCR-DGGE can be also used to rapidly checkthe diversity of the bacterial community after cultivationin liquid or solid and specific or nonspecificculture media. Briefly, after colony counting has beenperformed, the colonies from the plates can be collectedin ‘‘bulks’’ and subjected to DNA extractionand PCR-DGGE analysis (Ercolini et al., 2001b).Consequently, a DGGE fingerprint can be obtainedfor each plate, each dilution, and culture medium.This method to investigate the cultivable microbialcommunity has been shown to have good potential infood microbiology. Firstly, it provides an alternativeto traditional tools for the identification of dominantspecies. Qualification of the dominant species couldbe achieved by sequencing the DGGE bands arisingfrom the patterns corresponding to the highest dilutionsin spite of the isolation of single coloniesfollowed by purification and biochemical identification.The bulk from countable plates can be thus usedto identify dominant microbial species from the foodmatrix as performed by Ercolini et al. (2003a) inanalysing the Stilton cheese. The advantage is that,while sequencing of partial 16S rDNA fragmentsrepresents a sound tool for identifying species, identificationof species using phenotypic methods such assugar fermentation patterns may sometimes be uncertain,complicated, and time-consuming, particularlywithin LAB, due to an increasing number of speciesthat vary in a few traits (Quere et al., 1997).