scholarly journals thyA as a Selection Marker in Construction of Food-Grade Host-Vector and Integration Systems for Streptococcus thermophilus

2004 ◽  
Vol 70 (3) ◽  
pp. 1858-1864 ◽  
Author(s):  
Yasuko Sasaki ◽  
Yoshiyuki Ito ◽  
Takashi Sasaki
Keyword(s):  
Thymidylate Synthase ◽  
Streptococcus Thermophilus ◽  
Lactobacillus Delbrueckii ◽  
Vector System ◽  
Selection Marker ◽  
Temperature Sensitive ◽  
Erythromycin Resistance ◽  
Food Grade ◽  
Food Applications ◽  
Double Crossover Event

ABSTRACT We constructed food-grade host-vector and integration systems for Streptococcus thermophilus by using a thymidylate synthase gene (thyA) as the selection marker. Two thyA genes, thyASt and thyALb , were cloned from S. thermophilus and Lactobacillus delbrueckii subsp. bulgaricus, respectively. Thymidine-requiring mutants of S. thermophilus were obtained after successive cultures in the presence of trimethoprim, and one of them, TM1-1, was used as the host. Food-grade vectors were constructed by using either thyASt or thyALb as the selection marker. Transformants of TM1-1 created by using these vectors were selected for thymidine autotrophy as efficiently as for erythromycin resistance. By using the host-vector system developed in this way, a foreign amylase gene (amyA) was expressed in TM1-1 and was also integrated into the chromosome by use of a temperature-sensitive integration vector constructed with thyALb as the selection marker via a double-crossover event. The results obtained show that thyA is an efficient and safe selection marker for S. thermophilus that is suitable for food applications.

scholarly journals Use of an α-Galactosidase Gene as a Food-Grade Selection Marker for Streptococcus thermophilus

2005 ◽  
Vol 88 (7) ◽  
pp. 2341-2347 ◽  
Author(s):  
S. Labrie ◽  
C. Bart ◽  
C. Vadeboncoeur ◽  
S. Moineau
Keyword(s):  
Streptococcus Thermophilus ◽  
Selection Marker ◽  
Food Grade

Production of Recombinant β-Galactosidase in Lactobacillus plantarum, Using a pSIP-Based Food-Grade Expression System

2014 ◽  
Vol 931-932 ◽  
pp. 1518-1523 ◽  
Author(s):  
Numphon Thaiwong ◽  
Siwatt Thaiudom ◽  
Dietmar Haltrich ◽  
Montarop Yamabhai
Keyword(s):  
Enzyme Production ◽  
Expression Vector ◽  
Expression System ◽  
Selection Marker ◽  
Expression Systems ◽  
Recombinant Enzymes ◽  
Food Grade ◽  
Food Industries ◽  
Specific Enzymatic Activity ◽  
Food Applications

Food-grade expression systems based on using food-grade microorganisms have been developed for the production of recombinant enzymes used in food applications. Lactic acid bacteria (LAB), especially Lactobacilli, have been widely used for various purposes in food and recognized as a promising host of food-grade enzyme production. In this study, the pSIP409 vectors, originally containing the erm gene, were used to replace this selection marker by the alr gene resulting in the production of the pSIP609 expression vector in L. planatarum. This vector could express high amounts of β-galactosidases, showing both high volumetric as well a specific enzymatic activity. Thus, the food-grade recombinant enzyme production in L. planatarum harboring pSIP609 was very fruitful and useful for food industries.

scholarly journals Application of the shsp Gene, Encoding a Small Heat Shock Protein, as a Food-Grade Selection Marker for Lactic Acid Bacteria

2003 ◽  
Vol 69 (8) ◽  
pp. 4408-4412 ◽  
Author(s):  
Hassan A. M. El Demerdash ◽  
Knut J. Heller ◽  
Arnold Geis
Keyword(s):  
Heat Shock ◽  
Small Heat Shock Protein ◽  
Functional Trait ◽  
Selection Marker ◽  
Erythromycin Resistance ◽  
Gene Encoding ◽  
Food Grade ◽  
Transformation Rates ◽  
Standard Deviations ◽  
Average Transformation

ABSTRACT Plasmid pSt04 of Streptococcus thermophilus contains a gene encoding a protein with homology to small heat shock proteins (A. Geis, H. A. M. El Demerdash, and K. J. Heller, Plasmid 50:53-69, 2003). Strains cured from the shsp plasmids showed significantly reduced heat and acid resistance and a lower maximal growth temperature. Transformation of the cloned shsp gene into S. thermophilus St11 lacking a plasmid encoding shsp resulted in increased resistance to incubation at 60°C or pH 3.5 and in the ability to grow at 52°C. A food-grade cloning system for S. thermophilus, based on the plasmid-encoded shsp gene as a selection marker, was developed. This approach allowed selection after transfer of native and recombinant shsp plasmids into different S. thermophilus and Lactococcus lactis strains. Using a recombinant plasmid carrying an erythromycin resistance (Emr) gene in addition to shsp, we demonstrated that both markers are equally efficient in selecting for plasmid-bearing cells. The average transformation rates in S. thermophilus (when we were selecting for heat resistance) were determined to be 2.4 × 104 and 1.0 × 104 CFU/0.5 μg of DNA, with standard deviations of 0.54 × 104 and 0.32 × 104, for shsp and Emr selection, respectively. When we selected for pH resistance, the average transformation rates were determined to be 2.25 × 104 and 3.8 × 103 CFU/0.5 μg of DNA, with standard deviations of 0.63 × 104 and 3.48 × 103, for shsp and Emr selection, respectively. The applicability of shsp as a selection marker was further demonstrated by constructing S. thermophilus plasmid pHRM1 carrying the shsp gene as a selection marker and the restriction-modification genes of another S. thermophilus plasmid as a functional trait.

scholarly journals Molecular Characterization of a Theta Replication Plasmid and Its Use for Development of a Two-Component Food-Grade Cloning System for Lactococcus lactis

2001 ◽  
Vol 67 (4) ◽  
pp. 1700-1709 ◽  
Author(s):  
Éric Émond ◽  
Richard Lavallée ◽  
Geneviève Drolet ◽  
Sylvain Moineau ◽  
Gisèle LaPointe
Keyword(s):  
Lactococcus Lactis ◽  
Resistance Mechanism ◽  
Laboratory Strain ◽  
Plasmid Profile ◽  
Selection Marker ◽  
Erythromycin Resistance ◽  
Food Grade ◽  
Cloning System ◽  
Two Component ◽  
Industrial Strains

ABSTRACT pCD4, a small, highly stable theta-replicating lactococcal plasmid, was used to develop a food-grade cloning system. Sequence analysis revealed five open reading frames and two putativecis-acting regions. None appears to code for undesirable phenotypes with regard to food applications. Functional analysis of the replication module showed that only the cis-actingori region and the repB gene coding for the replication initiator protein were needed for the stable replication and maintenance of pCD4 derivatives in Lactococcus lactis. A two-component food-grade cloning system was derived from the pCD4 replicon. The vector pVEC1, which carries the functional pCD4 replicon, is entirely made up of L. lactis DNA and has no selection marker. The companion pCOM1 is arepB-deficient pCD4 derivative that carries an erythromycin resistance gene as a dominant selection marker. The pCOM1 construct can only replicate in L. lactis iftrans complemented by the RepB initiator provided by pVEC1. Since only the cotransformants that carry both pVEC1 and pCOM1 can survive on plates containing erythromycin, pCOM1 can be used transiently to select cells that have acquired pVEC1. Due to the intrinsic incompatibility between these plasmids, pCOM1 can be readily cured from the cells grown on an antibiotic-free medium after the selection step. The system was used to introduce a phage resistance mechanism into the laboratory strain MG1363 of L. lactisand two industrial strains. The introduction of the antiphage barrier did not alter the wild-type plasmid profile of the industrial strains. The phenotype was stable after 100 generations and conferred an effective resistance phenotype against phages of the 936 and c2 species.

scholarly journals Identification of the Propionicin F Bacteriocin Immunity Gene (pcfI) and Development of a Food-Grade Cloning System for Propionibacterium freudenreichii

2007 ◽  
Vol 73 (23) ◽  
pp. 7542-7547 ◽  
Author(s):  
Dag Anders Brede ◽  
Sheba Lothe ◽  
Zhian Salehian ◽  
Therese Faye ◽  
Ingolf F. Nes
Keyword(s):  
Selection Marker ◽  
Multiple Cloning Site ◽  
Propionibacterium Freudenreichii ◽  
Expression Plasmid ◽  
Physiochemical Properties ◽  
Food Grade ◽  
Cloning System ◽  
Immunity Gene ◽  
Membrane Bound ◽  
High Level

ABSTRACT This report describes the first functional analysis of a bacteriocin immunity gene from Propionibacterium freudenreichii and its use as a selection marker for food-grade cloning. Cloning of the pcfI gene (previously orf5 [located as part of the pcfABC propionicin F operon]) rendered the sensitive host 1,000-fold more tolerant to the propionicin F bacteriocin. The physiochemical properties of the 127-residue large PcfI protein resemble those of membrane-bound immunity proteins from bacteriocin systems found in lactic acid bacteria. The high level of immunity conferred by pcfI allowed its use as a selection marker for plasmid transformation in P. freudenreichii. Electroporation of P. freudenreichii IFO12426 by use of the pcfI expression plasmid pSL102 and propionicin F selection (200 bacteriocin units/ml) yielded 107 transformants/μg DNA. The 2.7-kb P. freudenreichii food-grade cloning vector pSL104 consists of the pLME108 replicon, a multiple cloning site, and pcfI expressed from the constitutive PpampS promoter for selection. The pSL104 vector efficiently facilitated cloning of the propionicin T1 bacteriocin in P. freudenreichii. High-level propionicin T1 production (640 BU/ml) was obtained with the IFO12426 strain, and the food-grade propionicin T1 expression plasmid pSL106 was maintained by ∼91% of the cells over 25 generations in the absence of selection. To the best of our knowledge this is the first report of an efficient cloning system that facilitates the generation of food-grade recombinant P. freudenreichii strains.

scholarly journals Encapsulation Through The Use Of Emulsified Microemulsions

2021 ◽  
Author(s):  
Ruby R. Rafanan
Keyword(s):  
Controlled Release ◽  
Continuous Phase ◽  
Pharmaceutical Products ◽  
Water Soluble ◽  
Scanning Calorimetry ◽  
Food Grade ◽  
X Ray ◽  
X Ray Scattering ◽  
Food Applications ◽  
Glycerol Monooleate

Emulsified microemulsions (EMEs), first described in detail in 2005 by the group of Garti, consist of a thermodynamically stable water-in-oil microemulsion phase (w1/o) further dispersed within an aqueous continuous phase (w2). These internally-structured w1/o/w2 dispersions are promising controlled release vehicles for water-soluble flavouring compounds, drugs and nutraceuticals. With a stable internal droplet structure, storage stability is improved over non-thermodynamically stable structured emulsions and may exhibit unique controlled release behaviour. Use of food-grade components allows for wider and safer applications in food and pharmaceutical products. In this thesis, a food-grade w1/o microemulsion consisting of glycerol monooleate, tricaprylin and water was dispersed in an aqueous (w2) phase by membrane emulsification and stabilized by a caseinate-pectin complex to produce w1/o/w2 EMEs. The resulting EME showed no signs of phase separation for weeks at room temperature. The microemulsion and EME were characterized by differential scanning calorimetry (DSC), cryo-TEM and small angle x-ray scattering (SAXS) to determine whether the microemulsion’s internal structure was maintained after emulsification. It was shown that EME droplets displayed ordering around the periphery consistent with some loss of microemulsion structure, but maintained the characteristic disordered microemulsion structure at the droplet core. Overall, this research demonstrated the feasibility of developing EME for possible applications in food and non-food applications.

scholarly journals Encapsulation Through The Use Of Emulsified Microemulsions

2021 ◽  
Author(s):  
Ruby R. Rafanan
Keyword(s):  
Controlled Release ◽  
Continuous Phase ◽  
Pharmaceutical Products ◽  
Water Soluble ◽  
Scanning Calorimetry ◽  
Food Grade ◽  
X Ray ◽  
X Ray Scattering ◽  
Food Applications ◽  
Glycerol Monooleate

Emulsified microemulsions (EMEs), first described in detail in 2005 by the group of Garti, consist of a thermodynamically stable water-in-oil microemulsion phase (w1/o) further dispersed within an aqueous continuous phase (w2). These internally-structured w1/o/w2 dispersions are promising controlled release vehicles for water-soluble flavouring compounds, drugs and nutraceuticals. With a stable internal droplet structure, storage stability is improved over non-thermodynamically stable structured emulsions and may exhibit unique controlled release behaviour. Use of food-grade components allows for wider and safer applications in food and pharmaceutical products. In this thesis, a food-grade w1/o microemulsion consisting of glycerol monooleate, tricaprylin and water was dispersed in an aqueous (w2) phase by membrane emulsification and stabilized by a caseinate-pectin complex to produce w1/o/w2 EMEs. The resulting EME showed no signs of phase separation for weeks at room temperature. The microemulsion and EME were characterized by differential scanning calorimetry (DSC), cryo-TEM and small angle x-ray scattering (SAXS) to determine whether the microemulsion’s internal structure was maintained after emulsification. It was shown that EME droplets displayed ordering around the periphery consistent with some loss of microemulsion structure, but maintained the characteristic disordered microemulsion structure at the droplet core. Overall, this research demonstrated the feasibility of developing EME for possible applications in food and non-food applications.

scholarly journals CHARACTERIZATION OF HAZELNUT MILK FERMENTED BY LACTOBACILLUS DELBRUECKII SUBSP. BULGARICUS AND STREPTOCOCCUS THERMOPHILUS

2018 ◽  
pp. 677-686 ◽  
Author(s):  
Ertan Ermiş ◽  
Rabia Güneş ◽  
İnci Zent ◽  
Muhammed Yusuf Çağlar ◽  
Mustafa Tahsin Yılmaz
Keyword(s):  
Streptococcus Thermophilus ◽  
Lactobacillus Delbrueckii ◽  
Lactobacillus Delbrueckii Subsp

scholarly journals Стан мнестичних процесів і рівень білків клітинної адгезії (NCAM) у гіпокампі щурів за умов введення S-аденозил-L-метіоніну та пре/пробіотиків на тлі медикаментозно-індукованого ураження печінки

2019 ◽  
Vol 13 (3) ◽  
pp. 166-174
Author(s):  
В.І. Жилюк ◽  
Г.О. Ушакова ◽  
Ю.В. Харченко ◽  
Д. В. Муравйова
Keyword(s):  
Lactobacillus Acidophilus ◽  
Lactobacillus Rhamnosus ◽  
Streptococcus Thermophilus ◽  
Lactobacillus Delbrueckii ◽  
Lactobacillus Delbrueckii Subsp

Мета дослідження – вивчення впливу S-аденозил-L-метіоніну, пре/пробіотиків та їхнього сумісного застосування на мнестичні процеси та рівень білків клітинної адгезії (NCAM) у гіпокампі щурів за умов тривалого введення рифампіцину та ізоніазиду. Дослідження проводили на 68 білих щурах-самцях лінії Wistar масою 180–220 г. Експериментальну модель токсичного медикаментозно-індукованого ураження печінки (МІУП) відтворювали шляхом повторних внутрішньошлункових (в/ш) введень ізоніазиду та рифампіцину у дозах 50 мг/кг і 86 мг/кг маси тіла відповідно протягом 28 діб. Тварин було розподілено на п’ять груп (n = 8 у кожній). І – інтактна, ІІ – контроль (МІУП). Щурам ІІІ групи протягом останніх 14 діб експерименту за 1 год до введення туберкулостатиків внутрішньом’язово вводили S-аденозил-L-метіонін у дозі 35 мг/кг. Щури IV групи в/ш отримували комбіновану терапію, що поєднувала пребіотик Лактулозу в дозі 2680 мг/кг і пробіотик, що містить 4 млрд активних клітин (КУО): Lactobacillus acidophilus, Lactobacillus rhamnosus, Streptococcus thermophilus, Lactobacillus delbrueckii subsp. Bulgaricus у дозі 1 КУО/кг. Тваринам V групи проводили потрійну комбіновану фармакотерапію з усіма препаратами, які застосовували в ІІІ і ІV групах у відповідних дозах. Ноотропну активність оцінювали в тесті умовної реакції пасивного уникнення (УРПУ) за умов введення скополаміну. Для оцінки впливу скополаміну на процеси навчання використані значення показників груп тварин (інтактні, n = 10; МІУП, n = 18), які попередньо не отримували цей препарат. Кількісний уміст NCAM визначали за допомогою інгібіторного методу імуноферментного аналізу з використанням моноспецифічних антитіл щодо NCAM (Аbcam, Англія) і відповідного очищеного білка як стандарту (Аbnovo, США) у цитозольній фракції гомогенату гіпокампа. Отриманий цифровий матеріал обробляли методом варіаційної статистики за допомогою програми статистичного аналізу StatPlus, AnalystSoft Inc. Версія 6 (http://www.analystsoft.com/ru/). Результати експериментів свідчать про те, що курсове введення S-аденозил-L-метіоніну покращувало процеси навчання за умов введення скополаміну, що проявлялося суттєвим збільшенням латентного періоду в 3,14 разу (Р = 0,027) порівняно з групою тварин з МІУП, а число тварин з набутою навичкою складало 62,5 % (Р = 0,007). При цьому за умов введення цього препарату спостерігалося зростання на 38,7 % (Р = 0,004) рівнів цитоплазматичної форми NCAM у гіпокампі, яке ймовірно мало компенсаторний характер. Характерно, що корекція стану мікробіоти кишечника також може чинити позитивний вплив на мнестичні функції за умов тривалого введення туберкулостатиків, однак за своїм ноотропним потенціалом комбінація Лактулоза/Йогурт поступалася S-аденозил-Lметіоніну, хоча рівні NCAM у гіпокампі зростали на 39,6 % (Р = 0,004) порівняно з групою тварин з МІУП. Слід зазначити, що сумісне застосування пребіотика та пробіотика з S-аденозил-L-метіоніном не супроводжувалося підвищенням ноотропної активності. Отримані дані експериментально обґрунтовують застосування насамперед S-аденозил-Lметіоніну, а також пре/пробіотиків як перспективних засобів корекції когнітивних порушень за медикаментозних гепатитів. Необхідні подальші дослідження щодо можливості комбінованого використання зазначених засобів за умов вказаної патології.

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