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Development of a real-time quantitat...

University of Delaware., Department of Animal and Food Sciences.

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Development of a real-time quantitative polymerase chain reaction method to quantify Lactobacillus buchneri in order to study its growth and effects in silages when added alone or in combination with Pediococcus pentosaceus.

Record Type:	Language materials, printed : Monograph/item
Title/Author:	Development of a real-time quantitative polymerase chain reaction method to quantify Lactobacillus buchneri in order to study its growth and effects in silages when added alone or in combination with Pediococcus pentosaceus./
Author:	Schmidt, Renato Jose.
Description:	169 p.
Notes:	Adviser: Limin Kung, Jr.
Contained By:	Dissertation Abstracts International69-09B.
Subject:	Agriculture, Animal Culture and Nutrition. -
Online resource:	http://pqdd.sinica.edu.tw/twdaoeng/servlet/advanced?query=3324457
ISBN:	9780549754077

Development of a real-time quantitative polymerase chain reaction method to quantify Lactobacillus buchneri in order to study its growth and effects in silages when added alone or in combination with Pediococcus pentosaceus.
Schmidt, Renato Jose.

Development of a real-time quantitative polymerase chain reaction method to quantify Lactobacillus buchneri in order to study its growth and effects in silages when added alone or in combination with Pediococcus pentosaceus. - 169 p.

Adviser: Limin Kung, Jr.

Thesis (Ph.D.)--University of Delaware, 2008.

The objective of these experiments was to develop a method to quantify Lactobacillus buchneri in silages in order to study its growth and effects in silages when added alone or in combination with Pediococcus pentosaceus. A unique set of primers based on a 16S rRNA fragment was developed to identify this bacterium in silage. The specificity of the primers was evaluated against different lactic acid bacteria and PCR products were only observed when using DNA from strains of L. buchneri as reaction templates. For the quantification, untreated samples of corn silage were inoculated with L. buchneri in the range of 10 3 to 108 cfu/g and the isolated DNA from these treated samples were used as standard for determining the number of L. buchneri (expressed as colony forming units per gram of silage) by real-time qPCR. A standard curve was constructed that allowed for the estimation of numbers of L. buchneri in silages. The technique was successfully used to quantify the population of L. buchneri in two cultivars of corn with or without inoculation. Thereafter, a series of experiments was conducted to investigate the growth of L. buchneri in silages and its effects on fermentation and aerobic stability. In two experiments, chopped alfalfa and chopped corn forage were left untreated or they were inoculated with L. buchneri alone or L. buchneri with P. pentosaceus and ensiled for various lengths of time. In alfalfa, inoculation with L. buchneri and P. pentosaceus together, resulted in a faster rate of fermentation during the very early stages of fermentation when compared to untreated alfalfa. This was characterized by a lower pH and a greater concentration of lactic acid. The epiphytic populations of L. buchneri could be detected in fresh, untreated alfalfa and corn but these populations never reached more than 6 log cfu/g, whereas in inoculated silages, numbers of L. buchneri peaked at approximately 9 log cfu/g of silage during the later stages of fermentation. Alfalfa and corn silages inoculated with L. buchneri alone and with P. pentosaceus had consistently higher concentrations of acetic acid and 1,2 propanediol, and undetectable populations of yeasts and molds during the later stages of ensiling than untreated silages. Aerobic stability was not determined in alfalfa but it was markedly improved in corn silages treated with inoculants containing L. buchneri. In a final experiment, when corn silage from five locations was inoculated with the same treatments previously described, numbers of L. buchneri (average of 6.70 log cfu/g of silage) were higher in four of five locations for inoculated silages in comparison with untreated silages (4.87 log cfu/g, tratment x location interaction). Silages inoculated with LB and LBC had higher silage pH and concentrations of acetic acid and 1,2 propanediol but lower concentrations of ethanol and water-soluble carbohydrates; but there was a treatment x location interaction for all these variables. The addition of L. buchneri 40788 alone or with P. pentosaceus improved silage fermentation and aerobic stability, but the effects were variable among locations, suggesting that unidentified factors in the field may alter the effectiveness of microbial inoculation. Collectively, these experiments showed that the numbers of epiphytic of L. buchneri in forages is very low and cannot reach high enough numbers to dominate the fermentation process. In contrast, when added to silages, L. buchneri grows slowly but eventually becomes a large proportion of the total lactic acid bacterial population. This slow growth relative to the growth of epiphytic lactic acid bacteria may be one reason why its effects are not seen during the very early stages of fermentation but its persistence during the later stages of storage is what defines its ability to improve the aerobic stability of silages.

ISBN: 9780549754077Subjects--Topical Terms:

1017857
Agriculture, Animal Culture and Nutrition.

Development of a real-time quantitative polymerase chain reaction method to quantify Lactobacillus buchneri in order to study its growth and effects in silages when added alone or in combination with Pediococcus pentosaceus.
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035 $a (UMI)AAI3324457
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100 1 $a Schmidt, Renato Jose. $3 1029905
245 1 0 $a Development of a real-time quantitative polymerase chain reaction method to quantify Lactobacillus buchneri in order to study its growth and effects in silages when added alone or in combination with Pediococcus pentosaceus.
300 $a 169 p.
500 $a Adviser: Limin Kung, Jr.
500 $a Source: Dissertation Abstracts International, Volume: 69-09, Section: B, page: 5110.
502 $a Thesis (Ph.D.)--University of Delaware, 2008.
520 $a The objective of these experiments was to develop a method to quantify Lactobacillus buchneri in silages in order to study its growth and effects in silages when added alone or in combination with Pediococcus pentosaceus. A unique set of primers based on a 16S rRNA fragment was developed to identify this bacterium in silage. The specificity of the primers was evaluated against different lactic acid bacteria and PCR products were only observed when using DNA from strains of L. buchneri as reaction templates. For the quantification, untreated samples of corn silage were inoculated with L. buchneri in the range of 10 3 to 108 cfu/g and the isolated DNA from these treated samples were used as standard for determining the number of L. buchneri (expressed as colony forming units per gram of silage) by real-time qPCR. A standard curve was constructed that allowed for the estimation of numbers of L. buchneri in silages. The technique was successfully used to quantify the population of L. buchneri in two cultivars of corn with or without inoculation. Thereafter, a series of experiments was conducted to investigate the growth of L. buchneri in silages and its effects on fermentation and aerobic stability. In two experiments, chopped alfalfa and chopped corn forage were left untreated or they were inoculated with L. buchneri alone or L. buchneri with P. pentosaceus and ensiled for various lengths of time. In alfalfa, inoculation with L. buchneri and P. pentosaceus together, resulted in a faster rate of fermentation during the very early stages of fermentation when compared to untreated alfalfa. This was characterized by a lower pH and a greater concentration of lactic acid. The epiphytic populations of L. buchneri could be detected in fresh, untreated alfalfa and corn but these populations never reached more than 6 log cfu/g, whereas in inoculated silages, numbers of L. buchneri peaked at approximately 9 log cfu/g of silage during the later stages of fermentation. Alfalfa and corn silages inoculated with L. buchneri alone and with P. pentosaceus had consistently higher concentrations of acetic acid and 1,2 propanediol, and undetectable populations of yeasts and molds during the later stages of ensiling than untreated silages. Aerobic stability was not determined in alfalfa but it was markedly improved in corn silages treated with inoculants containing L. buchneri. In a final experiment, when corn silage from five locations was inoculated with the same treatments previously described, numbers of L. buchneri (average of 6.70 log cfu/g of silage) were higher in four of five locations for inoculated silages in comparison with untreated silages (4.87 log cfu/g, tratment x location interaction). Silages inoculated with LB and LBC had higher silage pH and concentrations of acetic acid and 1,2 propanediol but lower concentrations of ethanol and water-soluble carbohydrates; but there was a treatment x location interaction for all these variables. The addition of L. buchneri 40788 alone or with P. pentosaceus improved silage fermentation and aerobic stability, but the effects were variable among locations, suggesting that unidentified factors in the field may alter the effectiveness of microbial inoculation. Collectively, these experiments showed that the numbers of epiphytic of L. buchneri in forages is very low and cannot reach high enough numbers to dominate the fermentation process. In contrast, when added to silages, L. buchneri grows slowly but eventually becomes a large proportion of the total lactic acid bacterial population. This slow growth relative to the growth of epiphytic lactic acid bacteria may be one reason why its effects are not seen during the very early stages of fermentation but its persistence during the later stages of storage is what defines its ability to improve the aerobic stability of silages.
590 $a School code: 0060.
650 4 $a Agriculture, Animal Culture and Nutrition. $3 1017857
650 4 $a Biology, Microbiology. $3 1017734
650 4 $a Chemistry, Analytical. $3 586156
690 $a 0410
690 $a 0475
690 $a 0486
710 2 $a University of Delaware. $b Department of Animal and Food Sciences. $3 1029904
773 0 $t Dissertation Abstracts International $g 69-09B.
790 $a 0060
790 1 0 $a Charley, Robert C. $e committee member
790 1 0 $a Emara, Marlene G. $e committee member
790 1 0 $a Joerger, Rolf D. $e committee member
790 1 0 $a Kung, Limin, Jr., $e advisor
790 1 0 $a Schmidt, Carl J. $e committee member
791 $a Ph.D.
792 $a 2008
856 4 0 $u http://pqdd.sinica.edu.tw/twdaoeng/servlet/advanced?query=3324457