Danisco United States Inc.

Waukesha, WI, United States

Danisco United States Inc.

Waukesha, WI, United States
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Andersen J.M.,Technical University of Denmark | Andersen J.M.,North Carolina State University | Barrangou R.,Danisco United States Inc. | Hachem M.A.,Technical University of Denmark | And 4 more authors.
Proceedings of the National Academy of Sciences of the United States of America | Year: 2011

Probiotic microbes rely on their ability to survive in the gastrointestinal tract, adhere to mucosal surfaces, and metabolize available energy sources from dietary compounds, including prebiotics. Genome sequencing projects have proposed models for understanding prebiotic catabolism, but mechanisms remain to be elucidated for many prebiotic substrates. Although β- galactooligosaccharides (GOS) are documented prebiotic compounds, little is known about their utilization by lactobacilli. This study aimed to identify genetic loci in Lactobacillus acidophilus NCFM responsible for the transport and catabolism of GOS. Whole-genome oligonucleotide microarrays were used to survey the differential global transcriptome during logarithmic growth of L. acidophilus NCFM using GOS or glucose as a sole source of carbohydrate. Within the 16.6-kbp gal-lac gene cluster, lacS, a galactoside-pentose-hexuronide permease-encoding gene, was up-regulated 5.1-fold in the presence of GOS. In addition, two β-galactosidases, LacA and LacLM, and enzymes in the Leloir pathway were also encoded by genes within this locus and up-regulated by GOS stimulation. Generation of a lacS-deficient mutant enabled phenotypic confirmation of the functional LacS permease not only for the utilization of lactose and GOS but also lactitol, suggesting a prominent role of LacS in the metabolism of a broad range of prebiotic β-galactosides, known to selectively modulate the beneficial gut microbiota.

Fan-Chiang H.-J.,Danisco United States Inc. | Wrolstad R.E.,Oregon State University
Journal of AOAC International | Year: 2010

Sugar and nonvolatile acid analyses were conducted on 52 samples of blackberries (Rubus spp), the objective being to develop a compositional database for evaluating authenticity and quality. ̊Brix ranged from 6.88 to 16.83, with a mean of 10.82. Titratable acidity ranged from 0.52 to 2.24 g citric acid/100 mL, with a mean of 1.35. Sucrose levels (range, 0-12.9%; mean, 4.6%) were highly variable. The overall glucose:fructose ratio ranged from 0.81 to 1.17, with a mean of 1.01. Malic, isocitric, lactoisocitric, citric, shikimic, and fumaric acids were identified, with succinic acid being present in some samples. Malic acid ranged from 5.2 to 35.3% of total acids (87.5-603 mgIIOO g), with a mean of 16.4% (280 mg/I 00 g). Isocitric acid ranged from 4.7 to 71.6%, with a mean of 34.7% (599 mg/100 g), and lactoisocitric acid ranged from 3.4 to 32.6% with a mean of 17.3% (293 mgIlOO g). Citric acid ranged from 1.3 to 80.2%, with a mean of 31.6 (572 mg/I 00 g). Shikimic, fumaric, and succinic were present in trace quantities. Two patterns of nonvolatile acid compositions were evident. Ten commercial blackberry juice samples were analyzed, and it was possible to determine whether they were Marion, Evergreen, or a mixture of the two from their acid profiles.

Hibberd M.C.,Danisco United States Inc. | Neumann A.P.,Danisco United States Inc. | Rehberger T.G.,Danisco United States Inc. | Siragusa G.R.,Danisco United States Inc.
Journal of Clinical Microbiology | Year: 2011

Clostridium perfringens is a ubiquitous and versatile pathogenic bacterium and is implicated in the etiology of the poultry diseases necrotic enteritis (NE) and poultry gangrene (PG). In this study, multilocus sequence typing was used to investigate genotypic relationships among 139 C. perfringens isolates from 74 flocks. These isolates had multiple disease, host, and environmental origins. The results indicated a polymorphic yet highly clonal population, with 79.6% of all isolates partitioning into one of six clonal complexes or two dominant sequence types, ST-9 and ST-31. The most prolific clonal complex, CC-1, contained 27.3% of all isolates and was not clearly associated with one particular disease. The subtypes CC-4 and ST-31 were highly associated with NE and represented 9.4% and 7.2% of the total isolates, respectively. No PG-associated and NE-associated C. perfringens isolates shared the same sequence type or clonal complex. NE-associated subtypes were more clonal and appeared more evolutionarily divergent than PG-associated subtypes, which tended to cluster in the more ancestral lineages alongside isolates from asymptomatic chickens and turkeys. Toxin gene screening identified cpb2 throughout these isolates and correlated the presence of netB with NE pathology. Previous investigations into the genetic basis of C. perfringens pathogenicity have focused on toxins and other variable genetic elements. This study presents the first sequence-based comparison of C. perfringens isolates recovered in clinical cases of PG and NE and demonstrates that niche specialization is observable in the core genomes of poultry-associated C. perfringens isolates, a concept with both epidemiological and evolutionary significance. Copyright © 2011, American Society for Microbiology.

Baker A.A.,Danisco United States Inc. | Davis E.,Danisco United States Inc. | Rehberger T.,Danisco United States Inc. | Rosener D.,Danisco United States Inc.
Applied and Environmental Microbiology | Year: 2010

Clostridium perfringens and Clostridium difficile are associated with scours in the neonatal piglet and are an economic concern in swine production. The objective of this study was to characterize the prevalence and diversity of C. perfringens and C. difficile isolates obtained from scouring neonatal piglets in a large integrated production system, as well as in smaller independently owned regional farms. Rectal swabs were collected from 333 pigs at 11 sites in an integrated swine production system and from an additional 180 pigs at 16 regional farms located throughout the Midwest. C. perfringens was isolated from 89.8% of the pigs swabbed at the integrated sites, and C. difficile was isolated from 57.7% of these pigs. Of the pigs from the regional farms sampled, 95.6% were positive for isolation of C. perfringens and 27.2% were positive for C. difficile. Toxigenic isolates were typed using random amplified polymorphic DNA (RAPD) PCR, and were placed in four dendrograms for C. perfringens and C. difficile populations isolated from the integrated sites and regional farms. Diversity indices showed that there was greater diversity in C. difficile populations and in populations isolated from the regional farms. A subset of isolates from the C. difficile dendrograms were further toxinotyped by amplification of the pathogenicity locus and subsequent digestion by HincII, AccI, and EcoRI. Of the 45 isolates typed, 44 were determined to be toxinotype V. The results of this study illustrate the diversity of C. perfringens and C. difficile isolates and the prevalence of these pathogens in swine production sites. Copyright © 2010, American Society for Microbiology. All Rights Reserved.

Sapranauskas R.,Vilnius University | Gasiunas G.,Vilnius University | Fremaux C.,Danisco | Barrangou R.,Danisco United States Inc. | And 2 more authors.
Nucleic Acids Research | Year: 2011

The CRISPR/Cas adaptive immune system provides resistance against phages and plasmids in Archaea and Bacteria. CRISPR loci integrate short DNA sequences from invading genetic elements that provide small RNA-mediated interference in subsequent exposure to matching nucleic acids. In Streptococcus thermophilus, it was previously shown that the CRISPR1/Cas system can provide adaptive immunity against phages and plasmids by integrating novel spacers following exposure to these foreign genetic elements that subsequently direct the specific cleavage of invasive homologous DNA sequences. Here, we show that the S. thermophilus CRISPR3/Cas system can be transferred into Escherichia coli and provide heterologous protection against plasmid transformation and phage infection. We show that interference is sequence-specific, and that mutations in the vicinity or within the proto-spacer adjacent motif (PAM) allow plasmids to escape CRISPR-encoded immunity. We also establish that cas9 is the sole cas gene necessary for CRISPR-encoded interference. Furthermore, mutation analysis revealed that interference relies on the Cas9 McrA/HNH-and RuvC/RNaseH-motifs. Altogether, our results show that active CRISPR/Cas systems can be transferred across distant genera and provide heterologous interference against invasive nucleic acids. This can be leveraged to develop strains more robust against phage attack, and safer organisms less likely to uptake and disseminate plasmid-encoded undesirable genetic elements. © 2011 The Author(s).

Namal Senanayake S.P.J.,Danisco United States Inc.
Journal of Functional Foods | Year: 2013

Green tea is one of the most popular and extensively used dietary supplement in the United States. Diverse health claims have made for green tea as a trendy ingredient in the growing market for nutraceuticals and functional foods. Green tea extract contains several polyphenolic components with antioxidant properties, but the predominant active components are the flavanol monomers known as catechins, where epigallocatechin-3-gallate and epicatechin-3-gallate are the most effective antioxidant compounds. Additional active components of green tea extract include the other catechins such as epicatechin and epigallocatechin. Among these, epigallocatechin-3-gallate is the most bioactive and the most scrutinized one. Green tea polyphenols are also responsible for distinctive aroma, color and taste. Green tea extract can also be used in lipid-bearing foods to delay lipid oxidation and to enhance the shelf-life of various food products. This review outlines the chemistry, flavour components, antioxidant mechanism, regulatory status, food applications, and stability of green tea extract in food. © 2013 Elsevier Ltd.

Bhaya D.,Carnegie Institution for Science | Davison M.,Carnegie Institution for Science | Davison M.,Stanford University | Barrangou R.,Danisco United States Inc.
Annual Review of Genetics | Year: 2011

Bacteria and archaea have evolved defense and regulatory mechanisms to cope with various environmental stressors, including virus attack. This arsenal has been expanded by the recent discovery of the versatile CRISPR-Cas system, which has two novel features. First, the host can specifically incorporate short sequences from invading genetic elements (virus or plasmid) into a region of its genome that is distinguished by clustered regularly interspaced short palindromic repeats (CRISPRs). Second, when these sequences are transcribed and precisely processed into small RNAs, they guide a multifunctional protein complex (Cas proteins) to recognize and cleave incoming foreign genetic material. This adaptive immunity system, which uses a library of small noncoding RNAs as a potent weapon against fast-evolving viruses, is also used as a regulatory system by the host. Exciting breakthroughs in understanding the mechanisms of the CRISPR-Cas system and its potential for biotechnological applications and understanding evolutionary dynamics are discussed. © 2011 by Annual Reviews. All rights reserved.

Barrangou R.,Danisco United States Inc. | Horvath P.,Danisco
Annual Review of Food Science and Technology | Year: 2012

Bacteria have been widely used as starter cultures in the food industry, notably for the fermentation of milk into dairy products such as cheese and yogurt. Lactic acid bacteria used in food manufacturing, such as lactobacilli, lactococci, streptococci, Leuconostoc, pediococci, and bifidobacteria, are selectively formulated based on functional characteristics that provide idiosyncratic flavor and texture attributes, as well as their ability to withstand processing and manufacturing conditions. Unfortunately, given frequent viral exposure in industrial environments, starter culture selection and development rely on defense systems that provide resistance against bacteriophage predation, including restriction-modification, abortive ection, and recently discovered CRISPRs (clustered regularly interspaced short palindromic repeats). CRISPRs, together with CRISPR-associated genes (cas), form the CRISPR/Cas immune system, which provides adaptive immunity against phages and invasive genetic elements. The immunization process is based on the incorporation of short DNA sequences from virulent phages into the CRISPR locus. Subsequently, CRISPR transcripts are processed into small interfering RNAs that guide a multifunctional protein complex to recognize and cleave matching foreign DNA. Hypervariable CRISPR loci provide insights into the phage and host population dynamics, and new avenues for enhanced phage resistance and genetic typing and tagging of industrial strains. Copyright © 2012 by Annual Reviews.

Sinkunas T.,Vilnius University | Gasiunas G.,Vilnius University | Fremaux C.,Danisco | Barrangou R.,Danisco United States Inc. | And 2 more authors.
EMBO Journal | Year: 2011

Clustered regularly interspaced short palindromic repeat (CRISPR) is a recently discovered adaptive prokaryotic immune system that provides acquired immunity against foreign nucleic acids by utilizing small guide crRNAs (CRISPR RNAs) to interfere with invading viruses and plasmids. In Escherichia coli, Cas3 is essential for crRNA-guided interference with virus proliferation. Cas3 contains N-terminal HD phosphohydrolase and C-terminal Superfamily 2 (SF2) helicase domains. Here, we provide the first report of the cloning, expression, purification and in vitro functional analysis of the Cas3 protein of the Streptococcus thermophilus CRISPR4 (Ecoli subtype) system. Cas3 possesses a single-stranded DNA (ssDNA)-stimulated ATPase activity, which is coupled to unwinding of DNA/DNA and RNA/DNA duplexes. Cas3 also shows ATP-independent nuclease activity located in the HD domain with a preference for ssDNA substrates. To dissect the contribution of individual domains, Cas3 separation-of-function mutants (ATPase+/nuclease- and ATPase-/nuclease+) were obtained by site-directed mutagenesis. We propose that the Cas3 ATPase/helicase domain acts as a motor protein, which assists delivery of the nuclease activity to Cascade-crRNA complex targeting foreign DNA. © 2011 European Molecular Biology Organization. All Rights Reserved.

Danisco United States Inc. and Spraying Systems Co. | Date: 2011-12-28

A system for treating a food product with a mold inhibitor via spray application in suspended form includes a conveyor for moving the food product as well as at least one upper spray nozzle above the conveyor and at least one lower spray nozzle located below the conveyor. In an embodiment of the invention, one or more end spray nozzles are provided to spray one or more vertical end surfaces of the food product. A fluid supply system for supplies pressurized fluid to the spray nozzles. In an embodiment of the invention, the fluid supply system includes a recirculating reservoir for maintaining the mold inhibitor in suspension. The spray guns are controlled via pulse width modulation such that the instantaneous flow rate and the time averaged flow rate are equal to respective predetermined allowable flow rates.

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