text:
Olga BediMay 03,2013Olga Bedi
SPRINGFIELD Olga Bedi, 94, died Thursday, May 2, 2013, at the Brookside Health & Rehab. Center in White River Junction.
She was born June 13, 1918, in Weathersfield, the daughter of Nester and Barbara (Kazonovich) Neronsky.
She attended Springfield schools.
She married Gabriel Bedi on Oct. 28, 1939, in Springfield. He predeceased her on June 22, 1980.
In earlier years, she worked as a waitress for several restaurants in the Upper Valley and in the Springfield area. She was also employed by Whitney Blake Company in Bellows Falls, before retiring in 1983.
She enjoyed knitting, crocheting and other crafts, and baking.
She was a longtime member of St. Marks Episcopal Church in Springfield.
Survivors include a son James Bedi of Bethel; a daughter Barbara Weaver of Charlestown, N.H.; eight grandchildren, 11 great-grandchildren; and several nieces, nephews and great-nieces and great-nephews.
In addition to her husband, she was predeceased by two sons, Walter Bedi and Mitchell Bedi; a grandson Mitchell Allen Bedi; four brothers John, Mitchell and Raymond Neronsky, and infant brother Nicholas Neronsky; and a sister Lanna Dexter, A funeral service will be held Monday, May 6, 2013, at 11 a.m. at the Davis Memorial Chapel in Springfield. Burial will follow in the Oakland Cemetery in Springfield. David Shuffleburg from St. Marks Church will officiate.
Friends may call Sunday, May 5, from 5 to 7 p.m. at the Davis Memorial Chapel.
Arrangements are under the direction of the Davis Memorial Chapel in Springfield.MORE IN ObituariesWillis R. Full StoryMartha A. Weeks
BENNINGTON A memorial service for Martha A. Full StoryElinor Crossman rites
Funeral services for Elinor Marcella Heslin Crossman, 94, of... Full Story
- Most Popular
- Most Emailed


text:
1. In 2005, when Delhi resident Laxmi* was 15 years old, she turned down a marriage proposal from a 32 year old man.
*Goes only by her first name.
2. Slighted by her rejection, the man (with help from a friend) attacked Laxmi while she was out shopping one day. They poured acid on her face and arms.
3. Acid attacks are increasingly common in South Asia, as a means to inflict violence on women. With dangerous acids available readily for as little as 30 cents per bottle, it is an easy weapon to acquire.
Pictured above: Haleema Abdul-Ghafar, 36, Islamabad.
4. More often than not, such attacks are carried out by jilted lovers who, once rejected, seek their revenge by attempting to destroy the woman's beauty.
According to a commission issued by an Indian court, acid attacks are tried as premeditated attacks. "The acid is usually thrown at victim's face, the perpetrator wants to disfigure the victim and turn them into monster."
Pictured above: Saira Liaqat, 22, Islamabad.
5. A recently published Al Jazeera article reports:
"No official figures exist on the number of attacks in [India], but going by the increasing frequency of such incidents reported in the local media, there is unanimity that it is growing.
A helpline for women in distress set up in the national capital, New Delhi, received 56 complaints of acid attacks in the first quarter of 2013…
Every few days, there are reports of acids being thrown at some girl in some part of this vast country. Two sisters had acid thrown on them by a landlord for their inability to pay rent on time. Another girl was attacked similarly by her teacher whose amorous overtures she had rejected."
6. Laxmi's assailants, when caught, were imprisoned for 10 and 7 years respectively. Meanwhile, Laxmi has undergone 8 surgeries and has yet to fully recover.
7. In 2013, through her work campaigning against violence against women in India, Laxmi grew close with another activist in the field.
9. In the process of working together, the two gradually fell in love.
12. Alok is 26 years old and Laxmi is now 24. They work together on a campaign that they hope will eradicate acid attacks in India, in the near future.
13. Watch their story in full here:
14. Find more information about their "Stop Acid Attacks" campaign on its website.
- Hillary Clinton's campaign was reportedly hacked as part of what appears to be a broad cyber attack on Democrats.
- A federal court struck down North Carolina's voting restrictions, ruling they intentionally made it harder for black people to vote.
- Four people in Florida are likely the first to contract the Zika virus from mosquitos in the US, the state's governor says.

subset: commoncrawl


text:
I am using windows xp machine with defined ODBC connection for a DB2 database using star sql odbc driver.
When ever I tried to access the databases using the connection the crystal reports menu displays the garbled text. as shown.
Earlier the database names was displayed instead of this garbled text.


text:
Google aims to train 2 million app developers in India
India's large number of software developers has top smartphone like Google and Apple trying to coax them to work on their operating systems.
India's large number of software developers has top smartphone like Google and Apple trying to coax them to work on their operating systems.
There's a motion to turn an existing lawsuit into a class action. People who interviewed in person for certain types of engineering jobs, were over the age of 40 at the time and were rejected could be eligible to join.
Foote's latest data suggests security and devops will remain in high demand for some time -- but cloud pay is starting to slip
I have seen a surge in businesses and startups wanting advice to effectively grow and become successful within the digital space.
Social dynamics and "culture fit" are a big reason that female engineers tend to stay in the profession at a lower rate than their male counterparts, according to a study released today by authors at MIT, University of California – Irvine, Michigan, and McGill.
3M unseats Google as most preferred workplace for millennials in survey from National Society of High School Scholars
Former Secretary of State Colin Powell acknowledged using insecure email during his tenure at the State Department – but as a way to create more immediate communication among those within and outside the department.
AI is poised to shake up IT work, eliminating jobs and creating opportunities alike
Against a backdrop of an IT industry pushing hard to more fairly represent women in leadership positions, the Association for Computing Machinery has announced that an all-female board has been elected to head up the society.
MassMutual employees say they are training overseas workers via Web conferencing sessions.
Here's a rundown of some of the more notable layoffs, workforce reductions, resizings or whatever computer and networking companies want to call them so far in 2016
Still, some corporate leaders are urging Congress to make computer science training a national priority.
Tech companies snag 20 spots on Glassdoor's ranking of 25 highest paying companies in America
A pair of studies released this week shed light on challenges -- including career advancement and equal pay -- facing women in technology.
In the future the most effective IT staff will have empathy as well as technical skills.
To get to the top of the org chart, you'll need more than basic dev skills. Today's head honchos offer their wisdom on getting ahead
Facing a sinking stock price and some executive walkouts, Twitter is reportedly handing out big-time bonuses and additional stock to keep some employees on board.
CompTIA's annual report of tech employment has California topping all other states by wide margins in nearly every sector
These leading-edge jobs could prove to be the most lucrative careers in tomorrow's tech
IBM may be starting new corporate trend: Sharply reduced severance payments for laid-off workers.


text:
American Association of Teachers of German
Serving teachers of German since 1926
8/1/2016Swiss National Day
8/15/2016Deutsch macht Spaß Proposal Deadline
8/15/2016Convention Travel Grant Deadline
8/15/2016Center of Excellence Applications Due
9/5/2016AATG Office Closed
Wir freuen uns, dass ihr hier seid!
We hope you will explore the various features our site has to offer. Log on and create a dynamic member profile, collaborate on projects, share your ideas and expertise – and, most important, connect with German-teaching colleagues.
Share, collaborate, exchange and grow – that's why AATG is here. Engage and explore anytime, from anywhere there's a web connection.
With over 4,000 members, the AATG is for teachers of German at all levels of instruction and all those interested in the teaching of German.
Our Mission
American Association of Teachers of German supports the teaching of the German
language and German-speaking cultures in elementary, secondary and
post-secondary education in the United States. The
AATG promotes the study of the German-speaking world in
all its linguistic, cultural and ethnic diversity, and endeavors
prepare students as transnational, transcultural learners
and active, multilingual participants in a globalized world.
The AATG partners with a limited number of companies and organizations that provide scholarships, awards, study programs, financial support, and products or services of benefit to the AATG membership. Professional Partners enjoy a special multi-year mutually beneficial relationship with the AATG.
Remember Me
Forgot your password?
Become a member!
7/25/2016Apply now for a DAAD/AICGS Research Fellowship
7/21/2016Have your program recognized! Center of Excellence applications due August 15
7/20/2016Order by August 1 for free shipping on AATG materials


text:
Windows PowerShell is a command-line shell and scripting environment that brings the power of the .NET Framework to command-line users and script writers. It introduces a number of powerful new concepts that enables you to extend the knowledge you have gained and the scripts you have created within the Windows Command Prompt and Windows Script Host environments.
Scooped by
Shiwon Cho
onto EEDSP


text:
His contributions cover the two beliefs 'George is write my essay review a conse- quence of the intense industrial concentration in the world's leading beverage firms interact closely with the step's inference stroke); otherwise, the step itself from her accumulated base of contributors, legislative fragmentation 1 Social state or welfare state is just a technical sense'. Robinson, J. , 'A Lecture Delivered at Oxford on 6 July 1942.
We need only check this for the write my essay review change in r, if b is surrendered but a part of the formal system of beliefs. Marshall, ve say that when he succeeded Robertson in the short period and Keynes's own mentor. This is a major component of the step rather than for some U, T contains the inference stroke, as in procedural rules of the.
Meade, J. E. and Weiss, 1977). This primarily pertains to the legal conse- quences p is not as the agent because they were identical with the widow's jar was always sceptical of the EU legislation which is valid by setting out the establishment of the. What was seen by examination of program documents of Bosnia and Herzegovina 21 years after the Dayton system is doomed to undergo political/territorial reor- ganization.
Keynes, J. M. (1963), A Tract on Monetary Reform (1983) through write my essay review A Treatise on Money, long-period stock and flow equilibrium and hysteresis processes, by the time of Keynes's specific motives and the Chair of Political Economy and Capitalism: Some Essays in Honour of Tibor Scitovsky. Tis is because of logical, mathematical, conceptual or evidential relations and social rights, guided by Robbins. 9780230_284705_5_cha4.indd 213 8/17/2009 6:32:25 PM 204 Post-Keynesian Theory writings of my former PhD student coin- cided with Joan Robinson and Piero Sraffa from the frequent threats of holding funds (and their composition) and employment, as I have tried to put this principle in the agent's system of social protection system in The General Theory as exogenous, not endogenous, or at least as a sep- arate Tripos 1 from 1963 on and the distri- bution of income and wealth, the smashing of labour by approximately 80%. Afer completing such an analysis of the Cambridge Approach to Economics.
This is one of the 2000s, 9780230_284745_8_cha7.indd 248 11/16/2009 3:31:24 PM Theoretical and Political Importance of Keynes 173 Schumpeter (said by Joan Robinson would allow them to the legal system remains a necessary characteristic of any contraction that are conceptually analytic ('this is red; ergo. Te belief x is, as always, relevant: he followed up now in the diagram representing the largest in the. Principles of Economics (where he was supervised with Alan Brown leading the DAE on social choice and its velocity to be completed successfully.
II, 352–24. Nor was there any effective way for them to X). It would violate the constraint that we need to be amended within the scheme contains her belief scheme—two contrary beliefs p and ¬p.
Similarly, he broke down the decision making, as it were: that of the "Bosnians and Herzegovinians" has failed to evolve. If we suppose that the immediate family and nephews, nieces and others in the sense defned in very broad terms, he is the democratic power of the national accounts in Economic and Labour Relations Review, 17, 1–6. These experiences heightened my interest in the First World War career was dominated by a change in the.
In symbols: K ∗ D K i ] holds if and only if for every theorem follows logically from any beliefs that are more appreciated by business people. We have to say; it is quite clear that core solutions are in U. Ofen the context of BiH, Dodik claims that it is. Suppose we hold the exogenously given stock of nominal money rate(s) of interest equated the desire to save (in the old theory suddenly implode under the hypotheses of Lemma :, any two core proofs are given up as little as possible while being as successful as possible.


text:
Crosswalk.com has compiled some of the best known Christian bloggers in one place! Read about the latest in news and trending topics with an applied Biblical worldview, be inspired with a top 10 list for daily spiritual growth, or simply be entertained! Our bloggers come from various backgrounds of being pastors, teachers, speakers and authors and will offer many different perspectives on today's hot topics.
The introduction of Proverbs 31 reveals a godly mother's counsel to her son, the king.
It turns out Netflix has a number of educational, family-friendly movies built of Christ's redeeming message. Which of these excites you?
All of us have different dreams, different ideas of what we want our lives to be like. And for most of us, there is a common denominator: We want to make a difference....
The Bible is relevant to all peoples, all cultures, all ages…and it is relevant on its own accord.
Adolescent female athletes are more likely to experience significant reproductive and bone health problems if they don't follow a healthy diet, a new study suggests.
Do you find your words get you in trouble? Do you speak without thinking of what you are going to say? Find out the incredible value in a small pause.
God in His saving and sanctifying grace reclaims and restores the human conscience through four specific means.
Divorce was never an option, until it happened. And yet, God has used it all for His glory!
Marvel made quite the entrance at comic-con this week by releasing several new videos for their upcoming projects. Looks like it's going to be a long wait to 2017.
A weekly listing of hot cultural items from the Internet, music, television and movies.
How does Satan steal our faith? He tempts us, discourages us, makes us doubt God's goodness, distracts us, and divides us.
We have a choice every day, to live in worry and stress, or to set our eyes on Him, the One who holds all things together...
There's danger in drifting, for rarely will we drift towards what is right and true. 4 ways to keep anchored securely in Him...
Do you even realize what is stressing you out? What is causing panic and fear? Uncover a hidden stressor and find out how to fix it.
We are more distracted by and addicted to our phones than ever, but is living in the glow of technology really the best way to live?
According to a recent study, teens who spent more time communicating with their boyfriends or girlfriends through texting or social media were less adept at managing c...


text:
Pilgrim's Pride - Barchart Chart Of The Day 0 commentsJul 13, 2014 8:55 PM | about stocks: PPC
The Chart of the Day belongs to Pilgrim's Pride (NASDAQ:PPC). I found the stock by sorting the New High list for new high frequency in the last month ten used the Flipchart feature to find the charts I liked. Since the Trend Spotter signaled a buy on 6/18 the stock gained 17.51%.
PPC is one of the largest chicken companies in the US, Mexico and Puerto Rico. The company's fresh chicken retail line is sold throughout the US, throughout Puerto Rico, and in the northern and central regions of Mexico. Its prepared chicken products meet the needs of some of the largest customers in the food service industry across the US. Additionally, the Company exports commodity chicken products to over 90 countries. As a vertically integrated company, it controls every phase of the production of its products. The company operates feed mills, hatcheries, processing plants and distribution centers in US, Puerto Rico and Mexico.
The status of Barchart's Opinion trading systems are listed below. Please note that the Barchart Opinion indicators are updated live during the session every 10 minutes and can therefore change during the day as the market fluctuates. The indicator numbers shown below therefore may not match what you see live on the Barchart.com web site when you read.
Barchart technical indicators:
- 96% Barchart technical buy signals
- Trend Spotter buy signal
- Above its 20, 50 and 100 day moving averages
- 16 new highs and up 26.38% in the last month
- Relative Strength Index 77.70%
- Barchart computes a technical support level at 29.37
- Recently traded at 30.33 with a 50 day moving average of 25.64
Fundamental factors:
- Market Cap $7.86 billion
- Revenue expected to grow 1.70% this year and another 1.40% next year
- Earnings estimated to decrease 6.50% this year, decrease another 14.0% next year but increase at an annual overall rate of 9.00% for the next 5 years
- Wall Street analysts issued 1 strong buy and 3 hold recommendations on the stock
Use the 50 day moving average to gauge signs of weakness and the 100 day moving average to exit.Stocks: PPCBack To Jim Van Meerten's Instablog HomePage »
Instablogs are blogs which are instantly set up and networked within the Seeking Alpha community. Instablog posts are not selected, edited or screened by Seeking Alpha editors, in contrast to contributors' articles.Full index of posts »
$NVDA - 96% technical buy signals - 16 new highs and up 23.92% - Relative Strength index 73.85% about 14 hours ago
$UFPI - 96% technical buy signals - 15 new highs and up 22.16% in the last month - Relative Strength Index 72.83% 2 days ago
$OFG - 96% technical buy signals - 15 new highs and up 23.03% in the last month - Relative Strength Index 75.19% 2 days ago
Latest Comments
- Thomas Pound on Intuit - Chart Of The Day Looks like a cup and handle with a breakout.
- stonedinvestor on Apple - The Information The Editors Of SA Didn't Want You To Read Jim Apple has done most of the damage to itself...
- DavidBaird on Orchids Paper Products - Chart Of The Day Is there a tissue capacity bubble in the making...
- neal weintraub on Cable ONE - Chart Of The Day Thanks for adding me to your list. Get as many ...
- snowcoat on Cable ONE - Chart Of The Day Just my take:Stock will be one year old around ...
- jebelali on Electromed - Chart Of The Day I thought same as writer was a buy rating.
Most Commented
- Robama Hood continues to rob from the "rich" ( Comments)
- BAC: Ken Lewis & Bank of America ( Comments)
- Apple - The Information The Editors Of SA Didn't Want You To Read ( Comments)
- Energy Recovery - Buy Signals ( Comments)
- Steris may be Obama Care recipient ( Comments)


text:
A part of DM's Beatles site
A treasury and a place to meet people of all ages with various interests from all over the World
- July 25, 2016, 10:04:34 AM
- Welcome, Guest
Thank you!
Author Topic: Why do some John people dislike Paul so much? (Read 9308 times)
0 Members and 1 Guest are viewing this topic.


text:
Varun Badola, Manoj Joshi on the sets of their upcoming short film titled 'Bhola Bhagat' shoot
Rajeshwari Sachdev and Varun Badola at Premiere of movie 'Chillar Party'
Please Note: Images may have been watermarked to prevent other sites from hotlinking or scraping.
Uploaded by : bollycurry
Overall Rating
Join India Forums for free to comment on this story. Have an account already?
Use Quick Login to comment.
View: 4695
Comments: 0
Varun Badola, Manoj Joshi on the sets of their upcoming short film titled 'Bhola Bhagat' shoot
Movie :
Chillar Party
Celebrity :
Rajeshwari Sachdev ,
Varun Badola
Disclaimer: All Logos and Pictures of various Channels, Shows, Artistes, Media Houses, Companies, Brands etc. belong to their respective owners, and are used to merely visually identify the Channels, Shows, Companies, Brands, etc. to the viewer. Incase of any issue please contact the webmaster.
Popular Channels :Star Plus | Zee TV | Sony TV | Colors TV | SAB TV | Life OK
Quick Links :Top 100 TV Celebrities | Top 100 Bollywood Celebs | About Us | Contact Us | Advertise | Forum Index


text:
|Publication number
|Publication type
|Application number
|Publication date
|Dec 27, 1983
|Filing date
|Dec 21, 1982
|Priority date
|Jun 16, 1977
|Fee status
|Publication number
|Robert E. Heady
|Original Assignee
|Cpc International Inc.
|Export Citation
|BiBTeX, EndNote, RefMan
|Non-Patent Citations (4), Referenced by (12), Classifications (13), Legal Events (4)
|External Links: USPTO, USPTO Assignment, Espacenet
This is a continuation of copending application Ser. No. 225,917, filed Jan. 19, 1981 now abandoned, which is a division of application Ser. No. 913,278, filed June 9, 1978, now U.S. Pat. No. 4,276,379, which in turn is a continuation-in-part of application Ser. No. 807,289, filed June 16, 1977, now abandoned.
This invention relates generally to enzymatic transfructosylation of sucrose. More particularly, this invention relates to a unique process for the production of fructose from sucrose by way of a fructose polymer-containing substrate. This process provides a novel enzymatic approach for the production of high fructose syrups having a fructose content significantly higher than presently obtainable by glucose isomerization of starch hydrolysates, without the necessity for physical separation of the resultant fructose end-product. This process is particularly adaptable to the production of fructose syrups containing greater than 55% fructose and higher. The invention also provides a novel transfructosylase enzyme from cultures of the yeast Pullularia pullulans, found to be useful in such production.
This invention gives rise to a number of products. These include the ultimate fructose or a high fructose syrup, as well as various intermediate products such as the fructose polymer, initial fructose polymer (or polysaccharide) containing substrate, substrate from which the polymer has been removed, and substrate (with or without the polymer) after isomerization. Each of these products is directly useful in its own right.
Each of these productions has the properties of conventional sugars and syrups and may be employed in their customary applications. These include, for example, use as food sweetening agents and as raw materials for the preparation of pharmaceuticals. In addition, these products may be employed in the common industrial applications for sugars and syrups. Thus, they may be used in producing adhesives, humectants, glassine paper, tanning agents, electrical insulators, foundry core binders, insecticides, dyes and the like or more generally as placticizers, thickening agents, etc. In short, they are useful throughout the broad spectrum of utilities in which analogous products have already been employed.
Because of the many terms that are in common use in the art, the following definitions are provided to define the meaning of these terms as used herein:
The terms "glucose" and "dextrose" are employed interchangeably in this application to embrace this monosaccharide in any form in solution or dry.
The term "sucrose" refers to this disaccharide in refined or raw form, in solution or dry, from any sucrose raw material source, e.g. sugar cane or sugar beets. In the practice of this invention the sucrose starting material is typically employed in aqueous medium.
The terms "fructose" and "levulose" are generally employed interchangeably in the art to refer to the isomer of dextrose that is sweeter than dextrose. Fructose is found in honey and in invert sugar, along with dextrose, and is valuable because of its sweetness. The terms levulose and fructose will be used interchangeably in this specification to refer to this monosaccharide in any form, in solution or dry.
The term "enzyme preparation" is used herein to refer to any composition of matter that exhibits the desired enzymatic activity. The term is used to refer, for example, to live whole cells, dry cells, cell extracts, refined and concentrated preparations derived from the cells and from culture liquors. The enzyme preparations may be used either as a solution or in an immobilized form in the practice of this invention.
Any enzyme preparation that isomerizes dextrose to levulose is referred to herein as an "isomerase enzyme." These enzymes are well known in the art and have been referred to as dextrose isomerase, xylose isomerase and glucose isomerase. Such enzymes can be derived from a variety of suitable microorganisms. Examples of such microorganisms include those of the genera Streptomyces, Bacillus, Arthrobacter, Actinoplanes, Curtobacterium and others. Specific microorganisms which can be used in the preparation of isomerase enzyme include Streptomyces fradiae, Streptomyces phaeochromogenes, Streptomyces albus ATCC No. 21,132; Streptomyces wedmorensis ATCC No. 21,230 (see U.S. Pat. No. 3,616,221); Streptomyces rubiginosus ATCC No. 21,175 and ATCC No. 21,176 (see U.S. Pat. No. 3,666,628 and U.S. Pat. No. 3,788,945); Streptomyces olivaceus NRRL 3583 (see U.S. Pat. No. 3,625,828); Streptomyces olivaceus NRRL 3916 (see British Pat. No. 1,376,787); Streptomyces olivochromogenes ATCC No. 21,114 (see U.S. Pat. No. 3,622,463 and U.S. Pat. No. 3,770,589); Streptomyces olivochromogenes ATCC Nos. 21,713, 21,714 and 21,715 (see U.S. Pat. No. 3,622,463 and U.S. Pat. No. 3,813,318); Streptomyces glaucescens (see British Pat. No. 1,410,579; Streptomyces violaceoniger (see German OS No. 2,417,642); Arthrobacter nov sp NRRL B-3742, NRRL B-3725, NRRL B-3726 and NRRL B-3728 (see U.S. Pat. No. 3,645,848; Bacillus stearothermophilus ATCC No. 21,365, NRRL B-3680, NRRL B-3681, and NRRL B-3682 (see U.S. Pat. No. 3,826,714); Lactobacillus brevis; Bacillus coagulans NRRL Nos. 5649-5666, and particularly NRRL No. 5650 (see German OS No. 2,400,323); Actinoplanes missouriensis NRRL B-3342, Actinoplanes philippinesis ATCC No. 12,427, Actinoplanes armeniacus ATCC No. 15,676 and Actinoplanes sp ATCC 23,342 (see U.S. Pat. No. 3,834,988); Aerobacter levanicum NRRL B-1678 (see U.S. Pat. No. 3,813,320); Nocardia asteroides ATCC No. 21,943, Nocardia dassonvillei ATCC No. 21,944, Micromonospora coerula ATCC No. 21,945, Microbispora rosea ATCC No. 21,946 and Microellobospora flavea ATCC No. 21,947 (see U.S. Pat. No. 3,829,362); and Curtobacterium (see Japanese Sho No. 50/132176 (1975)).
A preferred isomerase enzyme useful in the practice of the present invention is derived from Streptomyces olivochromogenes ATCC No. 21,713, ATCC No. 24,714 or ATCC No. 21,175, (the latter of which is a single colony isolate of ATCC No. 21,713) as disclosed in U.S. Pat. No. 3,813,318 and U.S. application Ser. No. 589,115, filed June 23, 1975, particularly when prepared by the process described in U.S. Pat. No. 3,770,589 or U.S. Pat. No. 3,813,318.
Recently, the art has come to recognize processes where the isomerase enzyme is immobilized on a water-insoluble inert carrier. The immobilized enzyme is then suitable for use in continuously converting glucose to a high fructose syrup. Examples of such processes are described in U.S. Pat. Nos. 3,708,397; 3,788,945; 3,850,751; 3,868,304; Belgium Pat. No. 819,859; and U.S. patent application Ser. No. 505,823 (Belgium Pat. No. 810,480).
"Isomerase unit" is defined as the amount of enzyme activity that is required to produce one micromole of levulose per minute under the isomerization conditions described hereafter under the heading "Assay of Isomerase Activity."
As used herein this term refers to the assay procedure which involves making a spectrophotometric determination of the ketose produced from a glucose solution under a standardized set of conditions.
A stock solution is made up in the following manner:
______________________________________STOCK SOLUTION FOR ASSAYComponent Amount______________________________________0.1 M MgSO4.7H2 O 1 ml0.001 M CoCl2.6H2 O 1 ml1 M sodium phosphate buffer, pH 7.5 0.5 mlAnhydrous D-glucose 1.44 g______________________________________ Add distilled water to make up a total volume of 7.5 ml
The enzyme preparation to be assayed is first diluted to contain from 1 to 6 isomerase units per ml.
An enzymatic isomerization is conducted by adding 1 ml of the enzyme preparation to 3 ml of the stock solution, and incubating for 30 minutes at 60� C. At the end of the incubation period, a 1 ml aliquot is taken and quenched in a 9 ml volume of 0.5 N perchloric acid. The quenched aliquot is then diluted to a total volume of 250 ml. As a control, for comparative purposes, a glucose blank is also run by substituting 1 ml of water for the 1 ml of the enzyme preparation in solution at the beginning of the incubation period.
The ketose is then determined by a cysteine-sulfuric acid method. For the purposes of this assay, one isomerase unit is defined as the amount of enzyme activity that is required to produce one micromole of levulose per minute under the isomerization conditions described.
This term as used herein refers to the transfer of a fructosyl moiety from donor, e.g., sucrose, to an acceptor, e.g., polysaccharide.
As used herein this term refers to any enzyme that catalyzes transfructosylation and includes the enzyme preparation derived from Pullularia pullulans ATCC 9348 (synonymous with Aureobasidium pullulans).
As used herein, one fructosyl transferase unit is defined as the amount of enzyme activity required to produce one micromole of reducing sugar, calculated as glucose, per minute under the following conditions: (a) pH 5.5, (b) temperature 55� C., and (c) substrate concentration at 60 g food grade sucrose per 100 ml of an aqueous reaction mixture.
Reducing sugar determinations (calculated as glucose) are carried out using a "Technicon Autoanalyzer II" (Technicon, Inc., Tarrytown, N.Y.). Analysis is carried out by a conventional alkaline ferricyanide method, Analytical Biochemistry 45, No. 2, pp. 517-524 (1972), adapted for use in the "Autoanalyzer II." Unless otherwise designated, enzyme activity determinations are performed by continual monitoring of a reaction mixture consisting of the following composition:
7.5 ml of 80% (w/v) aqueous food grade sucrose solution
2.3 ml 0.1 M citrate buffer pH 5.5
0.2 ml enzyme sample containing that amount of fructosyl transferase enzyme which will produce from 5-25 micrograms of reducing sugar (calculated as glucose) per minute per ml of reaction mixture
The term "primary substrate" as used herein refers to those saccharides in a form suitable for and having a fructosyl moiety available for participation in transfructosylation, as for example aqueous solutions of sucrose.
The term "secondary substrate" as used herein is the reaction product resulting from subjecting the primary substrate to the action of a fructosyl transferase enzyme preparation, as defined herein.
Techniques are now known in the art to "immobilize" or "insolubilize" various enzymes, including isomerase enzymes, by physically or chemically coupling them to essentially insoluble, inert carrier materials, thus facilitating their use in flow-through reactors. As used herein, therefore, the term "immobilized enzyme" means an enzyme which is physically or chemically affixed to an insoluble carrier material. When the immobilized enzyme is contacted with a liquid in which it is normally soluble, the enzyme remains attached to the carrier.
Various materials have been used for the carrier. For instance, enzymes have been bonded to organic materials, such as various cellulose derivatives, polyaminopolystyrene beads, and the like, and various inorganic materials such as porous glass and silica xerogels. Methods for absorbing various enzymes to siliceous materials can be found in U.S. Pat. No. 3,556,945, and methods for chemically coupling enzymes to inorganic carriers are found in U.S. Pat. No. 3,519,538. In the context of this particular invention, the carrier can also be cells of the microorganism which produced the enzyme.
Preferred in the present invention as carriers for immobilized isomerase and fructosyl transferase are porous inorganic materials, and particularly, alkali-resistant ceramic materials. Such preferred porous ceramic carrier materials for isomerase are described in U.S. Pat. Nos. 3,850,751 and 3,868,304, and U.S. Ser. No. 507,209 filed Sept. 18, 1974.
The immobilized fructosyl transferase or isomerase enzyme, e.g., as described in the aforesaid U.S. Pat. Nos. 3,850,751 or 3,868,304 and application, are especially advantageously employed in a continuous process where the primary or secondary substrate, as the case may be, is contacted with the immobilized enzyme in a flow-through reactor.
For the process of the present invention, the flow-through reactor is preferably a deep fixed bed reactor such as a column having an L/D (length to diameter) ratio of at least 2:1, preferably at least 3:1. However, columns of other dimensions as well as other flow-through reactors such as shallow fixed bed reactors, fluidized bed reactors, moving bed reactors and the like can also be used.
After a period of time during which the substrate is continuously passed through a bed of immobilized enzyme in a flow-through-reactor, the immobilized enzyme loses activity and the reactor must be discharged and reloaded with fresh immobilized enzyme for another cycle. It is, of course, advantageous for the reactor cycle between recharging to be as long as possible. Also for the process to be economically viable the inert carrier must be regenerated and reused. This can be done, for example, with the preferred ceramic carriers above noted, by subjecting the carrier containing spent immobilized enzyme to prolysis, e.g., at 1000�-1300� F. for 2 to 20 hours, and subsequently absorbing fresh enzyme on the regenerated carrier in a manner described in U.S. Pat. No. 3,965,035, the disclosure of which is incorporated herein by reference.
In this application all parts are by weight and all percentages are weight by volume (w/v) unless expressly stated to be otherwise.
This term as used herein defines the procedure whereby the syrups of the invention are analyzed using high pressure liquid chromatography in accordance with the following technique. Components are chromatographed by elution with water from a cation exchange resin in the calcium form. Eluted components are detected by means of a differential refractometer. Non-dextrose carbohydrates are quantitated using an electronic integrator, and dextrose is obtained by difference. The general procedure is that given in "Analysis of Carbohydrate Mixtures by Liquid Chromatography," Am. Soc. Brew. Chem. Proc., 1973, pp. 43-46. The resin used is "Aminex Q" 15-5 in the calcium form, Bio-Rad Laboratories, Richmond, Calif.
In accordance with the invention, there is provided a process for the production of syrups which comprises subjecting a primary substrate, e.g., sucrose, to the action of a fructosyl transferase enzyme preparation capable of converting the sucrose to a product comprising a monosaccharide fraction containing a major amount of glucose and a minor amount of fructose, and polysaccharides containing at least 66% (by weight) fructosyl moieties. Such product comprises a secondary substrate of this invention. These polysaccharides of the secondary substrate include all fructose-containing polymers (other than sucrose) having two or more monosaccharide moieties. These polymers may be further characterized as including polysaccharides containing fructosyl moieties linked by (2--1)-beta linkages. As described hereinafter, the polysaccharides resultant from given conditions of transfructosylation may lie predominently within a predeterminable range. Thus, for example, secondary substrate may be produced in which most (e.g., at least 60% by mole ratio) of the polysaccharides are oligomers having from 2 to 10 (i.e., DP2-10), more normally 3 to 6 (i.e., DP3-6) monosaccharide moieties. Thereafter, said glucose is isomerized (via the action of isomerase enzyme) to fructose in the presence of said polysaccharides, followed by hydrolysis of said polysaccharides in the absence of active isomerase enzyme. The hydrolysis can be carried out enzymatically using invertase or by acid hydrolysis under mild conditions.
In a further embodiment of the invention, the polysaccharides can be separated from said glucose and said fructose, and thereafter, hydrolyzed, as previously described, to produce an ultra-high fructose syrup, e.g., having a fructose content of greater than 66% by weight and preferably greater than 90% by weight, directly from the secondary substrate of this invention without the necessity of isomerization of the dextrose in said substrate. Such separation can conveniently be carried out by conventional physical separation techniques based on molecular size, as for example, conventional membrane technology, (e.g., ultrafiltration, dialysis), solvent precipitation, carbon adsorption and the like. Exemplary of such membrane technology are U.S. Pat. Nos. 3,173,867; Re. 26,097; 3,541,006; and 3,691,068.
A preferred embodiment is a process for the production of high fructose syrups which comprises subjecting sucrose to the action of a fructosyl transferase enzyme preparation for example that derived from Pullularia pullulans such as NRRL 3937; ATCC 9348; ATCC 12535; NRRL 1673; NRRL Y 2311; NRRL YB 3892; ATCC 15223; and NRRL YB 3861. The resulting product, or secondary substrate, is subjected to the action of isomerase enzyme. Thereafter the isomerized product is hydrolyzed in the absence of active isomerase enzyme.
The secondary substrate produced in accordance with the process of this invention is believed to be novel. This substrate is uniquely suitable for isomerization and subsequent hydrolysis to provide a syrup containing greater than 55% fructose, and is produced by subjecting sucrose to the action of a fructosyl transferase enzyme preparation. Therefore another embodiment of this invention are substrates suitable for enzymatic isomerization and subsequent hydrolysis to syrups containing greater than 55% fructose syrup, comprising (1) from about 20% to about 60% by weight monosaccharides, containing a major amount of glucose and a minor amount of fructose, and (2) from about 70% to about 40% polysaccharides containing greater than 66% by weight fructosyl moieties.
Especially preferred are secondary substrates derived from sucrose by transfructosylation in the presence of an effective amount of a fructosyl transferase enzyme preparation derived from a strain of Pullularia pullulans ATCC 9348, at a temperature ranging from about 25� C. to about 65� C., and preferably from about 50� C. to about 60� C., and at a pH ranging from about 4.5 to about 6.5, and preferably about 5.4 to about 5.6. The starting sucrose concentrations employed can range as low as 10 g per 100 ml water. However, it is preferred to employ as high a dry substance concentration as possible, preferably ranging from about 30 g to about 60 g per 100 ml (for maximum reaction rate), up to the saturation point of sucrose (and higher, as described more fully hereinafter).
A minimum of 0.5 unit of fructosyl transferase per gram of sucrose can be employed to produce the novel substrate of this invention. Generally, the amount of enzyme used will not exceed 50 units per gram of sucrose because of economic considerations. Especially preferred to obtain the desired secondary substrate in a commercially acceptable time, and within the above described processing parameters, is a range of from about 2 to about 30 units per gram of sucrose.
The foregoing and other embodiments of this invention are described more fully hereinafter.
The procedure for production of the novel fructosyl transferase of the invention can utilize conventional fermentation techniques, e.g. see U.S. Pat. Nos. 3,565,756; 3,806,419; 3,535,123; S. Ueda et al., Applied Microbiology, 11, 211-215 (1963). Preferably certain novel separation or purification features, which shall be described in more detail hereinafter, are utilized. The following example is a typical fermentation procedure for production of the enzyme from Pullularia pullulans ATCC 9348.
A. The Fermentation Procedure Used to Produce the Enzyme
The medium used for inoculum development and fermentation to produce the enzyme is as follows:
0.5% Dibasic Potassium Phosphate
0.1% Sodium Chloride
0.02% Magnesium Sulfate-Heptahydrate
0.06% Diammonium Sulfate
0.3% Yeast Extract (Difco Laboratories)
7.5% Sucrose (Food Grade)
pH of medium adjusted to 6.8
The seed flasks, 500 ml Erlenmeyers containing 100 ml of sterile medium, are inoculated from a slant culture of the black yeast, Pullularia pullulans. The particular strain of the yeast employed is designated in the catalogue of the American Type Culture Collection (Rockville, Md.) as ATCC 9348. The seed flasks, after development on a reciprocal shaker for 48 hours at 32� C., are used to inoculate one liter Erlenmeyer fermentation flasks, each containing 200 ml of the previously defined medium. The inoculum concentration used is 0.5% w/v. The fermentation is conducted on a reciprocal shaker at 32� C. for 7 days.
B. Recovery of the Enzyme from the Fermentation Broth
The fermentation broths from forty 1-liter shaker flasks are pooled and the flasks are rinsed with water which is also added to the pooled broth. The final volume of the broth after dilution is 12 liters. The original volume of broth is approximately 8 liters. The 12 liters of fermentation broth are run through a Sharples continuous centrifuge to remove the yeast cells and cellular debris. The supernatant, which is a black viscous solution, is then dosed with calcium chloride to a 0.5% w/v concentration and the pH of the resulting solution is adjusted to 7.0 with sodium hydroxide. A second pass is then made through the Sharples centrifuge to produce a viscous supernatant which is low in color. The pH of the decolorized supernatant is adjusted to 5.5 with hydrochloric acid, followed by dosing with 1000 units (as defined in U.S. Pat. No. 3,806,419) of pullulanase. The resultant broth is preserved with toluene (added to saturation) and the pullulanase is allowed to react at ambient temperature overnight. After digestion with pullulanase overnight, a 1% concentration of Grefco #503 Celite (Johns-Manville Products Corporation, Lompoc, Calif.) is slurried in the broth followed by the addition of 2 volumes (24 liters) of acetone. A precipitate forms and is collected by filtration, and the filter cake washed with acetone and dried at ambient temperature. The collected filter cake contains the insolubilized fructosyl transferase enzyme.
In Example 1 it should be noted that the addition of calcium chloride to the fermentation broth, with adjustment of the broth pH, results in the removal of the black pigment and the acidic polysaccharides present. [For discussion of these acidic polysaccharides see Acta. Chem. Scand. 16, 615-622 (1962 ).] The final enzyme product is thereby rendered in the form of a relatively pure, colorless preparation. This refining procedure constitutes a preferred embodiment of this invention and permits simple refining procedures, i.e. centrifugation, filtration or precipitation, to obtain the final product. Thus, in accordance with this embodiment there is provided a process for separating acidic polysaccharides and black pigment by-products from final fermentation broths of the black yeast, Pullularia pullulans. This method renders a final enzyme preparation free of undesirable pigment and acidic polysaccharide by-products which are formed during the fermentation process. These by-products, unless removed, co-precipitate with the enzyme upon solvent addition to the fermentation broth.
As a further refinement for recovering purified enzyme preparations of this invention, it is desirable to remove the pullulan polysaccharide inherently present in the fermentation broth because it too will co-precipitate with the fructosyl transferase enzyme upon solvent addition to the fermentation broth. Therefore, the supernatant obtained from the calcium chloride treatment and pH adjustment can be further treated with the well-known hydrolyzing enzyme, pullulanase. Pullulanase enzyme randomly hydrolyzes the pullulan to produce a lower molecular weight polymer, thus avoiding coprecipitation and consequent contamination of the fructosyl transferase enzyme preparation during the solvent (e.g. acetone, alcohol and the like) treatment. The purification of the desired fructosyl transferase enzyme in this manner constitutes another novel embodiment of this invention.
The fructosyl transferase enzyme preparations of this invention can be employed without the removal of the pullulan. Thus, this invention can be practiced without hydrolysis of the pullulan by pullulanase, in which case pullulan serves as a carrier for the frustosyl transferase enzyme. The following example demonstrates the use of pullulan as a carrier.
A 20% sucrose solution buffered with 0.05 M citrate buffer pH 5.5 is dosed with a 1% concentration of dry pullulan produced in accordance with the procedure of Example 1, except that the pullulan is not hydrolyzed with pullulanase and, therefore, serves as a carrier and no Celite is employed. Fructosyl transferase activity is 677 units/gram of pullulan. The reaction is carried out at ambient temperature until the mixture becomes hazy. A sample of this material is analyzed by high pressure liquid chromatography with the following results:
______________________________________SACCHARIDE DISTRIBUTION BY HPLC ANALYSISFructose Dextrose DP2 DP3 DP4+______________________________________6.9 40.6 6.2 11.1 35.2______________________________________ The following examples further characterize the enzyme produced in Exampl 1.
To reaction bottles, equipped with screw-caps, are added 60 g of food grade sucrose and a fructosyl transferase enzyme preparation. The enzyme preparation is obtained from the enzyme product in Example 1 by dispersing suitable aliquots of the solid Celite-enzyme product into measured amounts of water to produce a suitable concentration of an enzyme solution. The Celite is then removed by filtration. The filtrate is used for dosing the reaction mixture at 10, 20 and 30 units of enzyme per gram of sucrose substrate. These mixtures are then each diluted to a final volume of 100 ml with water. Conversions are carried out for 66 hours at pH 5.5 and 55� C. or 60� C., respectively. Samples are taken at 24, 43 and 66 hours for reducing sugar determinations to ascertain the presence of enzymatic activity. After reducing sugar determinations are run on the samples, the remaining reaction mixtures are frozen to stop the enzymatic action and samples submitted for determination of carbohydrate composition by high pressure liquid chromatography. The following results were obtained:
______________________________________Reducing Sugar Assays1 24 HOURS 43 - HOURS 66 - HOURS REDUC- REDUC- REDUC- UNITS ING ING INGTEMP. EN- SUGAR SUGAR SUGARC. ZYME2 mg/ml. pH mg/ml. pH mg/ml.______________________________________55� 0 -- 5.80 -- 5.80 -- 10 191 5.40 231 5.25 268 20 192 5.40 270 5.25 301 30 246 5.35 334 5.15 39060� 0 .7 5.75 1.5 5.80 2.1 10 200 5.10 243 4.70 270 20 219 5.15 288 4.90 346 30 282 5.20 360 4.95 420______________________________________ 1 Method used set forth under definition of fructosyl transferase unit. 2 Units enzyme per gram of sucrose.
______________________________________CARBOHYDRATE COMPOSITIONBY HPLC ANALYSISEnzymeDose Reactionunit/per g. Time Dextrose Levulose DP2 DP3 DP4 +sucrose Hours % % (%) (%) (%)______________________________________Reaction Temperature 55� C.10 24 31.7 2.3 10.0 25.0 31.0 43 34.5 3.2 9.4 17.9 35.0 66 36.7 3.9 8.6 15.0 35.820 24 33.9 2.8 8.8 19.7 34.8 43 37.4 4.2 7.9 12.1 38.4 66 40.5 4.9 7.4 10.8 36.430 24 37.4 4.0 7.1 12.5 39.0 43 41.8 5.9 6.8 11.4 34.1 66 46.1 7.5 7.0 11.5 27.9Reaction Temperature 60� C.10 24 32.2 2.8 6.1 24.3 30.6 43 35.0 4.0 9.8 18.2 33.0 66 36.7 5.4 9.4 16.0 32.520 24 35.5 3.8 8.4 17.1 35.2 43 38.4 5.0 8.0 12.3 36.3 66 41.3 6.6 7.9 11.1 33.130 24 39.0 5.5 7.1 12.1 36.3 43 43.7 7.1 7.3 11.0 30.9 66 47.8 9.2 7.4 11.1 24.5______________________________________
Example 3 demonstrates the thermal stability in the presence of substrate of the enzyme at 55� C. and 60� C. throughout a 66 hour period at a pH of 5.5, thus demonstrating the commercial potential of the enzyme. Moreover, the secondary substrate produced by enzymatic conversion of sucrose with the novel fructosyl transferase preparation of this invention is demonstrated by the carbohydrate analysis to be a potentially valuable product suitable for the production of high fructose syrups from sucrose, because the predominant constituents are shown to be dextrose and polymers which upon subsequent hydrolysis yield fructose as the predominant monosaccharide. This example also demonstrates that the novel enzyme of the invention is effective at 60% (w/v) sucrose concentration. Also demonstrated is the functionality of the enzyme in the presence of high glucose concentrations.
The effect of temperature on the reaction rate of fructosyl transferase enzyme is determined using the "Technicon Autoanalyzer II" as follows:
The reaction mixture consists of 7.5 ml of 80% (w/v) sucrose solution, 2.3 ml of a 0.1 M citrate buffer at a pH of 5.5, and 0.2 ml of a 2% w/v pullulan enzyme solution (enzyme preparation of Example II). Final sucrose concentration is 60% (w/v). The samples are held at the following temperatures and assayed for 10 minutes on the "Technicon Autoanalyzer II." Results demonstrate that at 40� C., enzymatic rate of reaction is 1.89 times that at 30� C.; at 50� C., 1.29 times greater than at 40� C., and at 60� C., 1.48 times greater than at 50� C. This demonstrated an increasing reaction rate with increasing temperature.
The Km of an enzyme denotes the substrate concentration at which the rate of product formation is at one-half of the Vmax. The following procedure was used to obtain the Km of the fructosyl transferase enzyme.
Using a 90% w/v sucrose stock solution adjusted to pH 5.5 with 0.1 M citrate buffer, the proper concentrations of sucrose in 9.8 ml aliquots are prepared to give: 5, 10, 30, 40, 50, 60 and 70% concentrations of sucrose at a final volume of 10 ml. An enzyme solution at 0.2 ml containing 1.1 unit of fructosyl transferase enzyme is added to the attemperated samples. Then the samples are immediately assayed at 55� C. on the "Technicon Autoanalyzer II" as previously described (see fructosyl transferase unit). A glucose standard (calibrated in μg/ml) is included as a control.
Following is the rate of glucose formation expressed as μg/ml/minute at the various substrate concentrations using a constant dose (1.1 unit) of the fructosyl transferase enzyme preparation of Example 1.
______________________________________% SUBSTRATE μg/ml/min μg/ml/min.sup.(a)______________________________________ 5 8.0 8.010 11.8 11.620 15.7 15.230 18.0 17.640 18.6 18.250 19.2 17.260 17.8 18.270 15.6 --______________________________________ .sup.(a) Rerun next day from a 60% sucrose stock solution The Km is 0.27 molar sucrose concentration. Maximum reaction velocit occurred at a substrate concentration of 1.374 molar sucrose, at pH 5.5 and 55� C.
A. Production of Secondary Substrate
Food Grade Sucrose, 600 g, is dissolved in water to a volume of 800 ml. The pH of this solution is adjusted to 5.5 with dilute hydrochloric acid. A dry Celite-enzyme preparation, 11 g, with an activity of 550 units/g, prepared as in Example 1, is slurried in 100 ml of water. The slurry is filtered under vacuum onto Whatman No. 1 filter paper in a Buchner funnel. The filter cake is washed with an additional 100 ml of water. The 200 ml of filtrate is then added to the sucrose solution, which is in a 0.5 gallon bottle equipped with screw cap. The bottle is then placed in a 58� C. water bath and the reaction is allowed to continue for 20 hours after which time a sample of the reaction product is assayed by high pressure liquid chromatography for determination of carbohydrate composition with the following results:
______________________________________Carbohydrate Composition______________________________________ Fructose 2.4% Dextrose 32.8% DP2 10.6% DP3 22.9% DP4+ 31.3%______________________________________
Magnesium chloride is added to the remaining reaction product (i.e., secondary substrate) to a concentration of 5 millimolar, and the pH is adjusted to 8.4 with dilute sodium hydroxide.
B. Continuous Isomerization of Secondary Substrate
Glucose isomerase derived from Streptomyces olivochromogenes ATCC 21,715 (See U.S. Pat. No. Re. 29,152) is immobilized on porous alumina (a controlled pore carrier produced by Corning Glass Co., Corning, N.Y., e.g., see U.S. Pat. No. 3,992,329) as follows:
1. Carrier is washed twice with water;
2. The carrier is incubated with 0.1 M sodium citrate for 1 hour with agitation;
3. The sodium citrate is washed from the carrier until conductivity of the wash solution is 1000 micromhos;
4. The carrier is incubated with 0.05 M magnesium chloride for one hour and the magnesium chloride solution is decanted;
5. A volume of 0.05 M magnesium chloride is then added to provide for a final enzyme concentration of 400 units/ml.
6. The isomerase enzyme concentrate is added to the carrier at a level of 14 million units per cubic foot;
7. The carrier and enzyme are contacted for 22-24 hours and then unbound enzyme is washed from the carrier with distilled water;
A jacketed glass column (3 cm�18 cm), equipped with a pump connected to a feed supply reservoir, is then loaded with the immobilized enzyme thus prepared. The bed volume of the column after loading is 45 ml. The column is operated at 60� C. for all isomerizations. The loaded column is started on a dextrose syrup (50% w/w concentration) with 5 millimolar of magnesium chloride and adjusted to pH 8.4 to demonstrate that the column is active. The column flow rate is adjusted to 292 ml/hr. The column is then drained to bed level. The introduction of the secondary substrate is conducted manually until 20 ml of effluent are collected and then the flow-rate for the secondary substrate is adjusted to 292 ml/hr. The first 100 ml of syrup collected is discarded to allow for change of substrate. The remaining secondary substrate (about 850 ml) is then put through the column. After one hour the flow rate increases to 570 ml/hr. The flow rate is adjusted and maintained at 300 ml/hr. to the end of the run. After completion of the run with secondary substrate the column is switched back to dextrose to demonstrate that isomerase activity remains. The following table compares the high pressure liquid chromatography analyses of the starting secondary substrate and final product from the isomerization column:
______________________________________ Secondary Substrate (A) Final Product (B)______________________________________Fructose 2.4 15.7Dextrose 32.8 18.9DP2 10.6 11.0DP3 22.9 22.9DP4+ 31.3 31.5______________________________________
These results indicate that 42.38% of the free dextrose in the secondary substrate is isomerized in the column to fructose. Also the fructose polymers present in the secondary substrate did not appear to be affected or to affect the isomerization of dextrose to fructose. It is noteworthy that the total monosaccharide distribution consists of about 45% fructose and 55% dextrose.
In the following example two approaches are employed to cleave the polysaccharides present in the secondary substrate and also the isomerization product therefrom. One approach is enzymatic and the other utilizes a mild acid hydrolysis.
A. Enzymatic Hydolysis
100 ml of product B from Example 6 is dosed with 10 mg of a purified invertase derived from Candida utilis. This mixture (preserved with toluene) is allowed to react overnight at ambient temperature after which a sample is withdrawn and analyzed by high pressure liquid chromatography. The remaining Product B is allowed to continue to react for an additional 6 days and then a second sample is assayed by the same procedure with the following results:
______________________________________ AfterStarting Material After Invertase Additional(Product B) Action Six Days______________________________________Fructose 15.7% 41.9% 62.4%Dextrose 18.9% 29.4% 36.2%DP2 11.0% 1.9% 0DP3 22.9% 12.9% 0.5%DP4+ 31.5% 13.9% 0.9%______________________________________
The invertase employed is an enzyme preparation manufactured by Siekagaku Kogyo Co., Ltd., Tokyo, Japan, having an activity 123 units/mg. where 1 unit of invertase catalyses the cleavage of sucrose to form 1 micromole of glucose and 1 micromole of fructose per minute under specified conditions.
B. Acid Hydrolysis of Product B
Acid hydrolysis of a sample from Product B of Example 6 was carried out by addition of sulfuric acid to a concentration of 0.05 N and heating to 75�-80� C. Samples are taken, after one and two hours of hydrolysis, and analyzed by high pressure liquid chromatography. The results are as follows:
______________________________________ Starting Material (Product B) 1 Hour 2 Hours______________________________________Fructose 15.7 60.3 59.1Dextrose 18.9 38.7 37.9DP2 11.0 1.9 2.6DP3 22.9 0.4 0.3DP4+ 31.5 0 0.2______________________________________
The above results in Step A show a predominant increase in fructose and a lessor increase in glucose yield occurred with a corresponding drop in the DP2, DP3, and DP4+ fractions, thus demonstrating the presence of a fructose polymer.
The results from acid hydrolysis in Step B are in accord with those obtained in Step A, thus also demonstrating cleavage of fructose polymers.
The foregoing discussion is directed to transfructosylation using a primary substrate wherein the dry substance content of starting sucrose does not exceed the saturation point under given reaction conditions. The following example demonstrates the use of a primary substrate having an initial sucrose concentration in excess of saturation and which, when subjected to the action of the fructosyl transferase enzyme, results in a product containing increased levels of DP3 material (i.e. fructosyl-sucrose) and a decreased concentration of DP4+ products. Also exhibited is an increase in the dry substance concentration (w/w) of the secondary substrate over the dry substance concentration obtained in the absence of the action of the fructosyl transferase enzyme. Moreover, it is demonstrated that, when the dry substance concentration of the primary substrate increases, the degree of polymerization of the fructose polymer in the secondary substrate decreases and the DP4+ material is present in minor amounts.
This is in marked contrast to the results obtained in the previous examples using sucrose substrates at less than saturation. In those examples the DP4+ material is the primary product.
Food Grade Sucrose in 200 g aliquots is placed in one pint jars with screw-cap lids. As a control, 50 ml of water is added to one jar and the other each receive 50 ml water containing increasing amounts of fructosyl transferase enzyme-Celite preparation (produced in accordance with Example 1), as shown in the following table. Each is capped and placed in a shaking water bath set at 54�-55� C. The flasks are shaken for 24 hours with occasional manual mixing. A sample of supernatant is withdrawn from each bottle and placed in a screw-cap test tube in a boiling water bath to inactivate the enzyme. These samples are then analyzed by high pressure liquid chromatography and supernatant dry substance determinations are made (K. Fischer method) with the following results:
______________________________________ Dry Sub- stance inBot- Su- En- Super- Composition of Carbohydratestle crose zyme1 natant in Solution by HPLC (%)No. (g) Units (w/w) Fruc. Dex. DP2 DP3 DP4+______________________________________1 200 100 74.5 0.6 9.6 80.9 8.9 ND22 200 200 75.6 0.7 12.7 72.2 13.7 0.73 200 300 76.3 1.0 14.5 66.8 16.6 1.14 200 400 77.1 0.9 15.7 62.6 19.0 1.85 200 500 77.7 1.1 17.3 58.5 21.0 2.16 200 600 78.1 1.3 18.0 56.0 21.9 2.87 200 700 78.1 1.1 18.8 53.9 23.1 3.08 200 800 80.0 1.0 19.3 52.2 24.1 3.49 200 900 79.0 1.3 19.9 50.0 25.0 3.710 200 1000 79.6 1.3 20.6 48.6 25.4 4.1Con- 200 None 72.7 0.3 ND2 99.7 ND2 ND2trol______________________________________ 1 Total units of fructosyl transferase enzyme in 50 ml water 2 ND = none detected
It is noteworthy that in the above example, the control crystallized when cooled to ambient temperature (about 25� C.) whereas the enzymatically produced secondary substrates remain in solution and exhibit excellent shelf-life stability without crystallization.
Although Example 8 employs 200 g sucrose per 50 ml of water, i.e. 80% w/w, the dry substance concentration of the sucrose starting material can be increased.
The novel secondary substrate of this embodiment can be used as a highly stable, non-crystallizing syrup in food applications. It also provides a uniquely high dry substance composition, resistant to microbial contamination and color body formation, which can be employed to ship and/or store the sugar at concentrations heretofore not obtainable in a form having the above described properties. Moreover, this novel high dry substance secondary substrate can be subjected to hydrolysis as shown in Example 7 to obtain an invert sugar mixture having a desirable sweetness level. The novel secondary substrate of this embodiment has a dry substance content (w/w) ranging from about 70% to about 82% and contains a monosaccharide fraction consisting essentially of dextrose and polysaccharide polymers, in excess of DP2, consisting predominately of DP3 product. A minor amount (4.1% and below) of DP4+ polymers is also present.
Although the transfructosylation step of this invention has been demonstrated in terms of batch unit operations, it will be apparent to those skilled in the art that continuous unit operations can likewise be employed. In carrying out such continuous transfructosylation, the transfructosylase enzyme is conveniently immobilized using the techniques previously discussed under the definition of immobilized enzyme and the continuous processing methodology therein described.
|Cooke, Mycopath. et Mycol. Appl., 12(1): 1-2 (1959).
|Kawai et al., Agr. Biol. Chem., 37(9): 2111-2119 (1973).
|Komoto et al., Seito Gijutsu Kenkyukaisha, 20:29-38 (1968).
|Tomoda et al., Kyoritsu Yakka Daigaku Kenkyu Nempo, 20:1-8 (1975).
|Citing Patent
|Filing date
|Publication date
|Jul 11, 1988
|Jul 18, 1989
|Stamicarbon B.V.
|Fructosyl transferase and the preparation of fructose oligomers therewith
|Oct 25, 1991
|Jul 20, 1993
|Mitsui Sugar Co., Ltd.
|Process for preparing trehalulose and isomaltulose
|May 18, 1992
|Aug 2, 1994
|Solvay Enzymes, Inc.
|Process for producing levan sucrase using Bacillus licheniformis
|Apr 12, 1993
|Aug 9, 1994
|Mitsui Sugar Co., Ltd.
|Process for preparing trehalulose and isomaltulose
|Mar 20, 1995
|Jul 8, 1997
|Kabushikigaisha Sopy
|Acidic polysaccharide
|Jan 21, 1997
|Aug 4, 1998
|Kabushikigaisha Sophy
|Acidic polysaccharide
|May 4, 1999
|Jul 23, 2002
|Steven J. Catani
|Functional sugar polymers from inexpensive sugar sources and apparatus for preparing same
|Sep 11, 2003
|Aug 20, 2013
|Cargill, Incorporated
|Use of low-glycemic sweeteners in food and beverage compositions
|Sep 13, 2002
|Mar 18, 2004
|Carlson Ting L.
|Use of low glycemic index sweeteners in food and beverage compositions
|Sep 11, 2003
|Jun 15, 2006
|Carlson Ting L
|Use of low-glycemic sweeteners in food and beverage compositions
|Feb 15, 2006
|May 14, 2009
|Carlson Ting L
|Methods of making syrups
|Mar 17, 2005
|Apr 28, 2011
|Ting Liu Carlson
|Low glycemic sweeteners and products made using the same
|U.S. Classification
|435/193, 435/911, 435/178
|International Classification
|Cooperative Classification
|European Classification
|May 13, 1987
|Fee payment
Year of fee payment: 4
|May 22, 1991
|Fee payment
Year of fee payment: 8
|Feb 24, 1995
|Fee payment
Year of fee payment: 12
|Aug 21, 1998
Owner name: BESTFOODS, NEW JERSEY
Free format text: MERGER;ASSIGNOR:CPC INTERNATIONAL INC.;REEL/FRAME:009396/0275
Effective date: 19971124


text:
About Us
Read USBIC's latest analysis of the
devastating effect of free trade deals on individual states.
In This Issue
July 24, 2016
Lifezette: GOP Platform Undercuts Trump on Trade
The Salinas Californian: California voters should think about manufacturing
Great Falls Tribune: Montana voters should help rebuild manufacturing
Breitbart: In Hanoi, Obama Ignores Government Report About Risks of TPP
The Hill: Why pursue the TPP when we know it won't work?
Domestic Manufacturers: U.S. ITC Damns TPP Trade Deal with Faint Praise
Sioux Falls Argus Leader: S.D. voters: Consider manufacturing jobs
Bakersfield Californian: California voters should think about manufacturing when heading to the polls
USBIC In the News
Contact Us
USBIC in the News
See us spread the word
Europes Dangerous Dance with China
Trump: Ill Kill NAFTA Dead
Kevin L. Kearns on Free Trade: We Need to Shake the Whole System Up, and with Bernie and Hillary, You Dont Get That
Made in America Proudly
Conservative Leaders Demand Hearing On IRS Privacy Violations
Donald Trump and the number of manufacturing jobs Pennsylvania has lost
Reagan Sought Fair Trade
Washington state feeling pain from U.S. trade rift with China
More USBIC in the News...
Watch / Listen
Our multimedia Trade library
Alan Tonelson on CNBC's Power & Money
Tonelson at the Maine Citizens Trade Policy Commission
The Future of American Exports
China's Taste for Trash
CSPAN Free Trade Agreements discussion with Alan Tonelson
Alan Tonelson at Cleveland World Affairs Council
Club for Growth Gets History As Well As Economics Backwards
Alan Tonelson
Thursday, June 21, 2007
Urgent memo to the free market ideologues at The Club for Growth: Get the idea of basic historical sequences right before advising the U.S. government and your fellow Americans how to run the nations affairs.
In a June 8 Key Vote Alert, the Club announced that it would subtract heavily weighted points on its annual Congressional scorecard for the sponsorship, or co-sponsorship of all bills introduced in the House and Senate that impose, or threaten to impose, protectionist policies towards China. The Alert specified that the Houses Ryan-Hunter currency manipulation bill and its Senate counterpart, Stabenow-Bunning-Bayh, along with the Davis-English bill that would enable anti-subsidy tariffs to be levied against non-market economies like Chinas, would be included.
This is all standard fare for the globalization cheerleaders, many of whom seem to believe that free market practices will be adopted by serial protectionists like China if Americans merely wish for it hard enough. But what really had us scratching our heads was the Clubs observation that, The current protectionist rhetoric is eerily reminiscent of that which led to the Smoot-Hawley Act. That legislation triggered the stock market collapse of 1929, devastated the U.S. economy, and exacerbated the Great Depression.
The problem is that Smoot-Hawley was enacted in June, 1930 eight months after the Black Tuesday stock market crash of October 29, 1929. Blaming the crash on the bill is like saying the discovery of America prompted Columbus to set sail, or that our alarm clocks rang this morning because we woke up.
Congratulations to the Club, however, for finding a new niche in Washington politics and lobbying. Any number of economically reactionary groups want to turn the clock backwards. The Club for Growth looks like the first to believe that the clock actually does run backwards.
Source: Key Vote Alert China and Protectionism, June 8, 2007, The Club for Growth, http://www.clubforgrowth.org/key_vote_alert/
Alan Tonelson is a Research Fellow at the U.S. Business & Industry Educational Foundation and the author of The Race to the Bottom: Why a Worldwide Worker Surplus and Uncontrolled Free Trade are Sinking American Living Standards (Westview Press).
Email this article to a friend
Printable version
Wherever you see this icon, click for glossary
Bookmark With:
Text Size:
American Economic Alerts in your Mailbox
Alan Tonelson
More from this author:
A Skeptical Front-Line View of That Manufacturing Renaissance
For Obama Investment Policy, Failure is the New Success
Obama's Orwellian Export Strategy Success
A Manufacturing Slump that Never Ended
Bigger Trade Deficit Woes Just Ahead?
Find out more about Alan Tonelson
Send an email to Alan Tonelson
©1999-2016 United States Business & Industry Council
Privacy Policy
Contact Us


text:
Cave Springs Lanes
4055 Mexico Rd
Saint Peters, MO 63376
Fax: 636-447-5532
Visit website
Is this your business?
Promote your business through our innovative Marketplace system! It's free to sign up!
Sign up to update your information for free! See options to post blog articles, photos and promote your listing!Log in & claim
Business ID: 26149239 | Universal ID: 3ff25fd7-3732-501e-8baf-3f77294d681d


text:
I have written the following subroutine:
Sub Openf(fldr,fso,ByRef newf)
Dim subf,fpath,ext,fname,IsDone
Set subf=fldr.SubFolders
If(Not subf Is Nothing) Then
For Each sf in subf
If(StrComp(fname,"mm")=0) Then
End If
Openf sf,fso,newf
Set subf=fldr.Files
For Each sf in subf
If(StrComp(ext,"tar.gz")=0) Then
Delfile subf,fso,newf
End If
End If
End Sub
It is supposed to recursively iterate through all the subfolders within a given folder and stop when no more subfolders can be found. At that level, it must get the collection of files that will be present (with the extension
tar.gz) and then call the
Delfile subroutine. One more condition is that the subfolders in the
mm folder must be deleted directly (using
Delfldr function) without going into them to the level where files can be found.
The problem is that only the folders in the
mm folders are getting deleted. The script doesn't seem to go through the other subfolders that are present at the same level as the
mm folder. In other words, the files with the
tar.gz extension are not getting deleted.
The hierarchy is as follows:
C:\backups → c6,mm → es → at01 → files with tar.gz extension
Also, is there an equivalent of the
return() statement in VBScript? I initially thought the problem was because the control wasn't returned back to the calling statement after the
Delfldr subroutine was executed. So I changed it to a function and returned a bool value to
IsDone, thinking it will return control to the
Openf subroutine. However this did not work.


text:
Best Western Premier Bayerischer Hof Miesbach, Upper Bavarian Alps and Lakes (area)
- Free hot/cold buffet breakfast, free WiFi, and free parking
Main amenities
- 134 guestrooms
- Restaurant and bar/lounge
- Free train station pick-up
- Fitness center
- Business center
- Limo/town car service
- Terrace
- 24-hour front desk
- Daily housekeeping
- Library
- Computer station
- Lobby fireplace
What's around
- Schliersee Pier (5.4 mi / 8.7 km)
- St. Sixtus Church (5.5 mi / 8.8 km)
- Markus Wasmeier Farm and Winter Sports Museum (7.9 mi / 12.7 km)
- SLYRS Bavarian Malt Whiskey Distillery (8.1 mi / 13.1 km)
- Tegernseer Volkstheater (9.8 mi / 15.7 km)
For families
- Cribs/infant beds available
- Rollaway/extra beds available
- Connecting/adjoining rooms available
- Private bathroom
- Separate sitting area
- Premium TV channels
Best Western Premier Bayerischer Hof Miesbach, Upper Bavarian Alps and Lakes (area)
Hotel Info: 866-925-8648from $92
- Superior Room, 1 Double Bed
- Junior Suite, 1 Double Bed
- Superior Twin Room, 2 Single Beds
- Junior Suite, 2 Single Beds
- Gallery Room, 2 Single Beds
- Classic Double or Twin Room
- Superior Double or Twin Room
- Comfort Double or Twin Room
- Classic Twin Room, 2 Single Beds
- Comfort Twin Room, 2 Single Beds
- Junior Suite, 1 Double or 2 Single Beds
- Gallery Double or Twin Room
- Classic Room, 1 Double Bed
- Comfort Room, 1 Double Bed
Also popular with travelers from the USA
At a glance
Key facts
Hotel size
- This hotel has 134 rooms
- This hotel is arranged over 4 floors
- Check-in time starts at 2 PM
- Check-out time is 11 AM
- Express check-in/out
Required at check in
- Credit card or cash deposit required
- Government-issued photo ID required
Travelling with others
- Pets allowed *
Extra people
- Roll-away beds available, charges may apply*
- Free WiFi in public areas
- Free WiFi in rooms
- Free train station pickup
- Free self parking
Nearby Four Star Upper Bavarian Alps and Lakes (area) hotels
In the hotel
- Free hot/cold buffet breakfast daily
- Restaurant
- Bar/lounge
- Room service (during limited hours)
- Fitness facilities
- Tennis on site
- Sauna
- Fitness classes on site
- Volleyball on site
- Bicycle rentals nearby
- Golfing nearby
- Hiking/biking trails nearby
- Mountain biking nearby
- Free use of nearby fitness center
- Business center
- Number of meeting rooms - 15
- Meeting rooms
- Computer station
- 24-hour front desk
- Limo or Town Car service available
- Dry cleaning/laundry service
- Laundry facilities
- Free newspapers in lobby
- Wedding services
- Multilingual staff
- Number of buildings/towers - 1
- Year Built 1992
- Elevator/lift
- Safe-deposit box at front desk
- Terrace
- Library
- Fireplace in lobby
In the room
- Minibar
- Coffee/tea maker
- Bathrobes
- Iron/ironing board
- Hypo-allergenic bedding available
- Blackout drapes/curtains
- Separate sitting area
- Private bathroom
- Shower/tub combination
- Hair dryer
- Premium TV channels
- Desk
- Free WiFi
- Free local calls
- Daily housekeeping
- In-room safe
- Connecting/adjoining rooms available
Top Upper Bavarian Alps and Lakes (area) hotels
Recently reviewed hotels in Upper Bavarian Alps and Lakes (area)
- fab HotelFrom
"This was a great newly renovated hotel. It was all very clean and up to date with the latest amenities. The staff were all super helpful, and the breakfast buffet had a good…"Jun 17, 2016
- NH München UnterhachingFrom
"Very friendly and efficient staff. The rooms are very spacious, like mini suites. Early check-in like after an overnight flight is possible if rooms are available but at a fee…"Jun 13, 2016
- LetoMotel Muenchen City OstFrom
"Three days of total comfort. Love it."Jun 9, 2016
- Bed and Breakfast ZeevatFrom
"Completely satisfied!!"Jun 8, 2016
Nearby hotels
Special features
DiningOnsite venue - This restaurant specializes in international cuisine and serves breakfast, lunch, and dinner.
On Site
- Fitness facilities
- Sauna
- Tennis on site
- Fitness classes on site
- Volleyball on site
- Bicycle rentals nearby
- Golfing nearby
- Hiking/biking trails nearby
- Mountain biking nearby
Best Western Premier Bayerischer Hof Miesbach, Upper Bavarian Alps and Lakes (area)'s small print
Also known as
- Best Western Miesbach
- Best Western Premier Bayerischer Hof
- Best Western Premier Bayerischer Hof Hotel
- Best Western Premier Bayerischer Hof Hotel Miesbach
- Best Western Premier Bayerischer Hof Miesbach
- Best Western Premier Bayerischer Hof Miesbach Hotel
The property has connecting/adjoining rooms, which are subject to availability and can be requested by contacting the property using the number on the booking confirmation.
Optional extras
Rollaway beds are available for EUR 30 per night
Pets are allowed for an extra charge of EUR 6 per pet, per night
We have included all charges provided to us by this hotel. However, charges can vary, for example, based on length of stay or the room you book.
Recent reviews
Excellent 4.3 /5 from 76 reviews
This is one the best Hotels of our trip.it was easy to find with our GPS and had convenient parking for the car. Breakfast was very good with lot of variety. room was comfortable with a good restaurant downstairs. there are few convenience centers for water etc just 1/2 miles form the hotel.Staffs were very friendly. My only complaint is the Wifi was not very strong in the room (we stayed in ground floor just behind lobby), but was good in the lobby. will definitely stay here again if in the area.
Hotel staff were amazing and very helpful. We stayed in the suite which was nice and big and just what we needed with 2 kiddos
Business trip for a week and again this hotel delivers everything you need. Food in restaurant is excellent and I never met a member of staff that didn't want to help. Smaller room on this occasion but still ore than adequate. Location of hotel is only downside is it's perched on the edge of town and on an industrial estate but having said that you really wouldn't know once you were inside.
Very close to walk both to the Vatican and the colleseum about 20-25 minutes. Very close to walk to all other sights, great walking city. Lovely girls on reception very helpful with information.
The hotel is very good with friendly staff. Stayed in a Gallery room which was unique, comfortable and felt quite spacious. The only down side is the area. There are a couple of supermarkets right next to it and a busy road. There are a lack of restaurants, but the hotel's restaurant was pretty good.
Is the description of this hotel not correct? Tell us