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Liver disease
Sep 18, 2014
Alcohol hotspots of England revealed: Manchester and Merseyside top the liver disease league, while people in the West County are the healthiest
Sep 15, 2014
Patients with Hepatitis C 'should have immediate access to potential life-saving therapies' - Irish Haemophilia Society
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How more than 160 liver patients have been barred from transplant operations for refusing to give up alcohol
Sep 17, 2014
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Sep 15, 2014
Generic Hepatitis C Drug Could Help 100 Million People Living with Disease
Sep 11, 2014
A Woman Told Her Boss About A Devastating Cancer Diagnosis. He Responded By Firing Her.
Sep 10, 2014
Medical breakthrough: Diagnosing disease could be a breath away
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What China Is Doing to Clear the Air and Fight Climate Change
Sep 01, 2014
The silent organ: Liver diseases sometimes have no symptoms
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New pill offers hepatitis C 'cure'


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BEST WESTERN Golden Sails Hotel
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- Continental Breakfast
- Swimming pool
- Room service
- Free High-Speed Internet
- Restaurant
- Fitness/Health center
TripAdvisor Reviews BEST WESTERN Golden Sails Hotel Long Beach
Travel Blogs from Long Beach
Venice, Long Beach and Huntington with KT
... We were all cheesing the whole time. All of us glad to be together finally. Venice beach was very different to the beaches we had seen before. The whole promenade was made up of shops selling all sorts of stuff illegal in most countries. The beach was beautiful and we sat chatting while people behind us got arrested much to our entertainment. After our parking time was up we got on the road to Long Beach were ...
Day 23 - San Diego to Long Beach and Queen Mary
... 8217; but lost it but have gained a Soviet submarine. It must be fun trading on the international attractions market; maybe they'll get a Maori Waka canoe next.
So, there's a self-guided audio tour and some attractions tours and a huge range of options and combinations of them so becoming informed to buy a ticket takes forever. We had to remind ourselves that this was run by the City so ...
Overdose on Shrimp Cures Jet lag!
First day in LA. To quote Joash, the longest day ever! We did start the 22/3 at home in Oz, final packing, toasted muffin for brekkie, itching to leave. Arrived at the airport with plenty of time to spare! Smoothie so happy to be married to time-keeper nut Ruthy! All the fam happy to be related to Smoothie who gets Virgin lounge as a perk of his endless trips away. Free hotdogs and beverages consumed by all. Again due to awesome Smoothie planning, had great seats on plane - 3 ...
Australia, East Coast
... our efforts. The following day we drove across to the suberb of Newport where were staying with my mom's very good friends from South Africa, Sharon & Gordon. Sharon has known me since I was born and I have very fond memories of both of them from I was very young. Newport isa short distance west of Melbourne city centre. Before we dropped by their house we drove into Melbourne city centre and visited the Melbourne Cricket Ground otherwise known as the M.C.G and did a tour of ...
Our roommates
... the top down, freezing cold, the wind in our hair and eye blowing cold air and ******* up our hair and filling our pores with road grime from the dirty city. we also get to listen to loud dirty rap with a heavy bass blasting our eardrums and we drive in circles and it takes all day. sometimes all we do is drive like this and we always regret saying "sure we'll come." ...we get a new black light in the mail every day ;)

subset: commoncrawl


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Los Agaves Restaurant
Quick service - we all received our meals within five minutes of placing our orders; delicious serve-yourself salsa bar with 7 different options; reasonable prices; food quality a step above the normal Mexican fare
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Santa Barbara Bowl0.63 mi from Los Agaves Restaurant19 reviews
A really great place to see a summer concert under the stars or on a sunny Sunday afternoon. We caught NPR's A Prairie Home Companion here, and it was tons of fun to listen to Garrison Keillor spin his Midwestern yarns and here San Diego bluegrass musicians play at this outdoor stone amphitheater.Popular withFamiliesLocalDesign
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Santa Barbara County Courthouse0.91 mi from Los Agaves Restaurant19 reviews
An architectural delight and free! Climb to the top for a breathtaking view of the city and some great photo ops. On the way up, check out the huge bells that chime every 15 minutes. Also, the murals and tile work are gorgeous! It's the prettiest building the city.Popular withHistoryLocalAdventure
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Santa Barbara Winery0.87 mi from Los Agaves Restaurant9 reviews
The best Jerry, the best!Popular withFoodiesLuxuryTrendsters
- 40.88 mi
Chase Palm Park0.88 mi from Los Agaves Restaurant5 reviews
A great family park with an old fashioned horse carousel, koi pond, kids play facilities and an outdoor stage.Popular withFamilies
- 50.85 mi
East Beach0.85 mi from Los Agaves Restaurant6 reviews
Fun and games at the most popular Beach in town. Local Artist sell their Art every weekend. Stearns Warf is a "must do" on everyones list that goes to Santa Barbara. Only a couple of years ago a lost whale mysteriously decided to stay in the Harbor to entertain all visitors for almost 2 weeks. Great on the Warf restuarants ...Popular withOutdoorFamilies
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The Santa Barbara Bowl Foundation0.63 mi from Los Agaves Restaurant4 reviews
Great concerts. Around the corner. Walk up and buy a ticket.Popular withLocalNightlifeFamilies
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Kunin wines0.94 mi from Los Agaves Restaurant3 reviews
Cute wine bar on the Urban wine trail! Try it out!Popular withFoodiesTrendstersBusiness
- 80.95 mi
Municipal Winemakers0.95 mi from Los Agaves Restaurant3 reviews
This place is about as unpretentious as you can get when it comes to a wine tasting environment. It's eclectic and laidback, with a definite urban vibe - which makes sense as it's part of Santa Barbara's Urban Wine Trail. This tasting room is my favorite one part of the Urban Wine Trail - both for the environment and the ...Popular withAdventure
- 90.9 mi
The Funk Zone0.9 mi from Los Agaves Restaurant2 reviews
a blend of high energy and relaxing pubs, breweries, wineries, restaurants and street art all within two blocks of the beach. a must when coming through Santa Barbara.Popular withDesignNightlifeFoodies
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Walk this street and check out the sweet Art!!Popular withLocalOutdoorBudget
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Funk Zone0.9 mi from Los Agaves Restaurant2 reviews
New place just up from the beach and the pier. Only really coming into its own in the last 2 years, it has become the cool place to go...About 8 wine tasting rooms, Figueroa Mountain Brewery, Lark Restaurant (great variety of interesting foods) and the Lucky Penny (good food, inexpensive, eat outside) pizza and salad place next door
- 120.88 mi
TONIC NIGHTCLUB0.88 mi from Los Agaves Restaurant2 reviews
One of the most popular nightclubs in DT Santa Barbara, Tonic is frequently crowded over the weekends. The nights I would usually go to Tonic would be Thursday night, since that is college night for most clubs downtown. Out of all the clubs in SB, Tonic definitely has the longest wait time. The club inside has a dance floor and ...Popular withAdventure
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Santa Barbara Historical Society Museum0.73 mi from Los Agaves Restaurant2 reviews
First, it's free (donations are encouraged). Second, the adobe building is atmospheric and makes you feel like you've stepped into Old California. Third, the exhibits are a mix of interesting painting and photography exhibits matched with the permanent collection of oddly fascinating historical artifacts in the back. Definitely worth seeing, though it'll only take you an hour or so.Popular withHistory
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This is a cool and obscure little state park. It's so obscure, you could be standing in it and not even know it. It's a neat little piece of California history.Popular withAdventureFamiliesHistory
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Shopping center in downtown Santa Barbara. Shopping and eateries and movie theater. It gets crowded and it's hard to find parking, so we don't like to come here often. But this is a good spot for people who are out of town to walk around an outdoor shopping mall built for tourists.
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Surfaholics unite with the top of the top in everything surf! Sweet shop... Check it out!Popular withOutdoorAdventure
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Extremely beautiful views of the mountains and the Pacific. Such an amazing outdoor venue that you can dress up for a wedding or any other event that you may want to host.Popular withOutdoor


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UFC officials on Tuesday admitted that company president Dana White breached protocol by pulling a judge at its self-regulated UFC Fight Night Macao on Saturday. read news >>
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Clerkenwell is best known for the Museum of the Order of St. John, St. John's Gate located on St. John's Lane. The Museum is at what was once the entrance to the medieval priory of the Order of St. John, in a Tudor gatehouse dating from 504A.D. In the 12th century The Knights Hospitaller was founded under the order to care for travel weary Crusaders. Today those places of care are called hospitals. The St. John's ambulance Brigade was started in 1877. Later the buildings were used for offices, a public house, a coffee shop, and housed the offices of "Gentleman's Magazine."
Today it stands as the offices of the British order of St. Johns. The museum traces the history of the building through a collection of books, armor, paintings, pharmacy jars and medical instruments--some quite grisly. Free admission includes a guided tour of the building, cemetery, and 12th century crypt. While admission is free, donations are requested to help with refurbishments and up keep. Clerkenwell is also the site of London's oldest church dating back to 1123. It is also where the socialist "London Patriotic Club" was established, although now the area is known more for traditional English restaurants and clubs.
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|Wishful Fields Archive Target Field History
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"You talk about the magic, the aura, but what really makes a stadium is the fans. Concrete doesn't talk back to you. Chairs don't talk back to you. It's the people who are there, day in, day out, that makes the place magic."
– Bernie Williams
Explore the Site
Here are 50 images chosen randomly from the 3042 found on this site. Click the image to be taken to the original post. A new list is created every 10 minutes.
An early concept for the pedestrial bridge. (Source: Ballpark Authority, RP)
Not from Moose's tour, but it's an image you need to see. (Click to enlarge greatly.)
Auxiliary scoreboard (note to TF principles: this is a very good idea)
The dish!
Panels arriving on flatbed trailers in front of the Twins' dugout.
Dan Kenney provided this alternate shot of a walkway behind the view level
Work beneath the scoreboard
The plate marker is just to the left.
This looks from the base of the stairs, behind the big pillars, toward the street.
Looking for some detail
Steps, skyway, and plaza intersect.
Seventh inning sing-along.
Let's be honest and say that this promenade, which will face the HERC plant, won't be the most exciting part of the streetscape. It has to be provided for circulation reasons, but there won't be much to see unless vendors and other attractions take root here.
Opening Day 2008 (By Currier & Ives)
Loading dock -- already in use!
Pile driving in progress
This is the Metropolitan Club as viewed from the future Ballpark Authority office space.
Flag poles, fencing, main entrance gates
The reverse angle shows that the signage will only partially obscure views from the top of the ramp. The wall is pretty high up there, so you'll need something to stand on, but it appears that this is one of the so-called "knotholes".
Looking across the plaza toward the main ticket area.
Ticket window at Gate 29/Carew
Now, THIS is just some guy who appears to be hanging out on the LRT tracks talking to himself.
Walkway construction is progressing
Scoreboard installation in progress
You are forgiven for wondering whether architect Tom Oslund is, in fact, a visitor from the future.
Here is a close-up of those funny little islands of seats (HRP View).
Now, why is there horse shit on the street next to Target Field? (I saw it in two places. Mounted police maybe?)
Then you turn around to this!
The view from the Penn Ave entrance to 394 (and all the way into town! Click to enlarge)
Welcome Visitors From
Two Men On (Accessiblity)
BPM - Ballpark Magic
BRT - Bus Rapid Transit
DSP - Dave St. Peter
FSE - Full Season Equivalent
FYS - Fake Yankee Stadium (see also: NYS)
HERC - Hennepin Energy Resource Company (aka the Garbage Burner)
HPB - Home Plate Box
HRP - Home Run Porch
LC - Legends Club
LRT - Light Rail Transit
MBA - Minnesota Ballpark Authority (will own Target Field)
MOA - Mall of America
MSFC - Minnesota Sports Facilities Commission (owns the Metrodome)
NYS - New Yankee Stadium
SRO - Standing Room Only
STH - Season Ticket Holder
TCFBS - TCF Bank Stadium
TF - Target Field
Selected Bibliography - Analysis
Selected Bibliography - Surveys
Selected Bibliography - Nostalgia


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Eugene Peni has always had an active lifestyle but he once tipped the scales at 162kg.
He says it was a combination of poor diet and a lack of knowledge about how to live healthily that did it.
Now his weight has dropped to 100kg and a lot of the people who once doubted he'd ever trim down are coming to him for advice.
He is happy to help and is working towards a diploma in sport fitness at Unitec so that he can do it professionally.
It is a big change for a man who had never thought a career in sports or physical activity was possible.
Things turned around when he discovered the Village Sports Academy founded by All Black legend Michael Jones.
A friend who tutored at the academy challenged him to join.
"That's a sports leadership academy and you don't really expect a big guy like I was to go into something like that," he says. "From there it was all about breaking a lot of barriers. Every person there was athletic and fit and I was an obese person and didn't think I should be in an environment like that."
Peni overcame his insecurities and knuckled down with the support and encouragement of his classmates and tutor.
It was not easy, especially the physical side of things, but he soon found his rhythm.
"For the first few months it was really hard, the physical stuff was a massive challenge and I was always last or at the back.
"But midway through is when a lot of things changed for me."
A low-carb diet that ruled out bread, rice and potatoes helped the weight fall off.
"I pretty much changed the whole lifestyle.
"Previously me in a gym would never happen, and the first time was really intimidating because as a really big person you think everyone is watching you and you think 'what am I doing here, I should just go back outside'.
"It wasn't until after I lost the weight that I realised the people who were staring at me weren't looking at me like I shouldn't be there, but more like it was inspiring and 'good on you'."
From there he realised he was "pretty good at losing weight" and the idea of making a career out of helping others in the same situation became a reality.
"My ultimate goal is to own my own gym and help people like I've been helped, but I'm a believer in needing smaller goals to reach that one so for now it's study and maybe personal training.
"But now I don't think so many things are impossible like I used to."
Village Sports Academy chairman Michael Jones says the course is designed to use sport to hook people into further education.
"Engaging young Maori and Pacific youth is an important part of the programme, but the course is really for everyone," he says.
- Central Leader
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AAI wins government contract worth up to $600M
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- Gary Haber
- Staff Reporter- Baltimore Business Journal
- Email | Twitter
AAI Corp., the Hunt Valley maker of unmanned drone aircraft, has won a government contract worth as much as $600 million, and nabbed part of a second giant contract.
AAI was awarded March 2 a three-year deal worth up to $600 million to supply unmanned drone aircraft and the workers to operate them for the U.S. Special Operations Command's Mid-Endurance Unmanned Aircraft System program. The contract is with the Special Operations Command at MacDill Air Force Base in Tampa, Fla.
AAI also will share a five-year contract worth of up to $874 million with two other companies to provide and support unmanned aircraft systems for the U.S. Department of Defense and other government agencies.
The contract, awarded on Feb. 29, is with the U.S. Naval Air Systems command at Patuxent River Naval Air Station. AAI is sharing the contract with Computer Sciences Corp., (NYSE: CSC), of Falls Church, Va., and Insitu Inc., of Bingen, Wash. The contract runs through February 2017.
As a result of the contracts, AAI plans to hire additional field service workers to operate aircraft systems at customer locations, said Sharon Corona, a company spokeswoman. The number of employees the company would hire has not yet been determined. There is no information at this time about the number of employees, if any, AAI would add in Hunt Valley, Corona said.
AAI, a division of Textron Inc. (NYSE: TXT), in January laid off 184 workers at its facilities in Baltimore County. Before the layoff, AAI employed about 1,650 people at several locations in Baltimore County. The company employs a total of about 2,400 people worldwide.
Gary Haber covers Banking, Finance, Insurance, Law
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Born out of the 120 Hour Film Festival at The Sherry Theater in Los Angeles, this film was completed in it's entirety in just 5 days.
Inspired by the work, life and death of Francesca Woodman.
Directed by Ciera Parrack
120 Productions INC
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A row over former justice secretary Elsie Leung Oi-sie's recent criticism of the legal profession intensified yesterday when she hit back at her critics, saying she was simply exercising her right to free speech.Saturday, 13 October, 2012, 12:16pm 3 comments
Access to papers sought in billionaire divorce4 Feb 2012 - 12:00am
The Secretary for Justice has joined the legal fray over the multibillion-dollar estate of Nina Wang Kung Yu-sum, taking High Court action to register an interest in the handling of the late tycoon's will.
The secretary lodged a caveat to the will in the High Court yesterday demanding that no disbursements be made from the estate without the secretary being informed.7 Jul 2007 - 12:00am
Former justice secretary Elsie Leung Oi-sie, now the deputy head of the Basic Law Committee, attends the inauguration of the Federation of Guangxi Community Organisations in Wan Chai yesterday. Formed by 16 groups of Guangxi natives in Hong Kong, the federation hopes to strengthen ties between the city and the autonomous region, which is part of the pan-Pearl River Delta area.19 Dec 2006 - 12:00am
The report 'Part-time judges' political ties backed' on Page A2 yesterday wrongly stated that the secretary for justice had no wish to amend guidelines that require full-time judges to stay out of politics. It should have referred to the chief justice, who issued the guidelines, not the justice secretary. We apologise for the error.26 Jul 2006 - 12:00am


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SKIRTS Interview w/ GodTube - Today's Christian Videos
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Marshawn Evans reflects back on some of her experiences that inspired her to write her new book, "SKIRTS in the Boardroom."
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Aaron Paul
- Real Name:
- Aaron Paul Sturtevant
- Date of Birth:
- August 27, 1979
- Hometown:
- Emmett, ID
- Resides in:
- Los Angeles
Aaron Paul made a small and humble entrance into life — he was born a month premature in his parents' bathroom — but thanks to his breakout role on arguably one of the greatest TV series in history, charisma and voracious Twitter following he is currently living life large and in charge. After working as an usher at the Universal Studios Movie Theatre in Hollywood in the late '90s and appearing as a contestant on "The Price Is Right" in 1998, he bulked up his résumé with one-off appearances on shows like "Beverly Hills, 90210," "Melrose Place" (Frat Boy #2), "Suddenly Susan," "The X-Files," "NYPD Blue," "CSI," "ER" and "Veronica Mars" as well as a few minor film roles like "K-PAX," "Van Wilder" and "Mission: Impossible III." He moved up to a recurring character with a first and last name on the short-lived "Point Pleasant" in 2005, but it wasn't until his multi-season arc on "Big Love" that viewers really started to know the actor's real name. In 2008, he landed "Breaking Bad." His profanity-happy character Jesse Pinkman was supposed to die in the first-season finale, but he had so impressed producers that they instead kept him around until the end in 2013. He was nominated for three Outstanding Supporting Actor Emmys for the drama. The two-time winner wed Lauren Parsekian in May 2013 and has been busy filming one film after another since "Bad" ended including "Need For Speed" and "Hellion."
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From Elle
From Crowd Ignite


text:
Compositions useful in the treatment of dietary disorders, and as dietary additives to provide a source of fiber and of short chain fatty acids, to reduce the level of serum cholesterol and as bulking agents in humans and animals, as well as methods of use therefor are described. The compositions and methods are based on whole β-glucans.
1. A dietary supplement for administration to mammals consisting essentially of intact hollow whole β-glucan in an amount sufficient to provide a source of fiber in the diet to aid in digestion, reduce serum cholesterol levels and enhance weight loss in said mammal.
2. A dietary supplement of claim 1 wherein the whole β-glucan has been chemically modified by treatment with acid.
3. The dietary supplement of claim 2 wherein the acid comprises acetic acid.
4. The dietary supplement of claim 1 wherein the whole β-glucan has been enzymatically modified by treatment with a β-glucanase enzyme.
5. The dietary supplement of claim 4 wherein the β-glucanase enzyme is laminarinase.
6. The dietary supplement of claim 1 wherein the whole β-glucan comprises altered β-glucan derived from yeast cells which have been genetically modified.
7. The dietary supplement of claim 1 which is a laxative.
8. The dietary supplement of claim 1 which contains about 5 to about 50 grams of whole β-glucan.
9. A dietary supplement for reducing the level of serum cholesterol in a human or animal consisting essentially of an amount of intact, hollow whole β-glucan sufficient to reduce the level of serum cholesterol in said human or animal.
10. A dietary supplement of claim 9 wherein the amount of whole β-glucan is from about 5% to about 8% by weight of total dietary intake.
11. A dietary supplement for reducing weight gain or enhancing weight loss in a human or animal consisting essentially of an amount of intact, hollow whole β-glucan sufficient to reduce weight gain or enhance weight loss in said human or animal.
12. The dietary supplement of claim 11 wherein the amount of whole β-glucan is from about 5% to about 8% by weight of total dietary intake.
13. A method of providing a source of fiber in a diet of a mammal consisting essentially of administering hollow, intact whole yeast β-glucan to the mammal.
14. The method of claim 13 wherein the whole yeast β-glucan is in a biologically acceptable carrier.
15. The method of claim 14 wherein the carrier is sterile saline or water, and the composition is administered orally or through a feeding tube.
16. A method of claim 14 wherein the whole yeast β-glucan is administered to the mammal as part of a complete nutritional food formulation.
17. A method of claim 13 wherein the bulking and digestibility of the fiber is altered by increasing or decreasing the relative proportion of β(1-3) and β(1-6) linkages.
18. The method of claim 13 wherein the amount of whole β-glucan administered is from about 5 to about 50 grams per day.
19. A method of providing a fecal bulking agent to a mammal consisting essentially of administering hollow, intact whole yeast β-glucan to the mammal.
20. The method of claim 19 wherein the whole yeast β-glucan is in a biologically acceptable carrier.
21. The method of claim 20 wherein the carrier is sterile saline solution or water, and the composition is administered orally or through a feeding tube.
22. A method of claim 20 wherein the whole yeast β-glucan is administered to the mammal as part of a complete nutritional food.
23. A method of providing a source of short chain fatty acids to a mammal consisting essentially of administering to the mammal an amount of hollow, intact whole yeast β-glucan sufficient to aid digestion and improve intestinal function.
24. A method of reducing total plasma and LDL cholesterol in a human or animal comprising administering to the human or animal an amount of hollow, intact whole yeast β-glucan sufficient to cause reduction in total plasma and LDL cholesterol in the human or animal.
25. A method of claim 24 wherein the whole yeast β-glucan is derived from a strain of S. cerevisiae.
26. The method of claim 24 wherein the amount of whole β-glucan administered comprises from about 5% to about 8% by weight of the total dietary intake of the human or animal.
27. The method of claim 24 wherein the whole yeast β-glucan is in a biologically acceptable carrier.
28. The method of claim 27 wherein the biologically acceptable carrier comprises cornstarch.
29. A method of claim 28 wherein the whole yeast β-glucan is contained in a chewable tablet.
30. A method of reducing weight gain or enhancing weight loss in a human or animal comprising administering to the human or animal an amount of hollow, intact whole yeast β-glucan sufficient to cause a reduction in weight gain or an increase in weight loss.
31. The method of claim 30 wherein the whole yeast β-glucan is derived from a strain of S. cerevisiae.
32. The method of claim 30 wherein the amount of whole β-glucan administered comprises from about 5% to about 8% by weight of the total dietary intake of the human or animal.
33. The method of claim 30 wherein the whole yeast β-glucan is in a biologically acceptable carrier.
34. The method of claim 33 wherein the biologically acceptable carrier comprises corn starch.
35. The method of claim 34 wherein the whole yeast β-glucan is contained in a chewable tablet.
36. A liquid formulation comprising a non-gelling aqueous containing about 30 g/L of hollow, intact whole glucan.
Certain natural fibers benefit digestive function, help prevent intestinal and colon cancer and act as fecal gastroenteral bulking agents. Generally, fibers are derived from grains, and are composed of polysaccharides with a range of structures. The role of fiber in health has become well recognized; however, its mechanisms of action are not completely understood.
Yeast cell walls are largely made up of the hydrophilic polysaccharide beta-glucan. The cell wall is primarily composed of a β(1-3)-linked glucose polymer with periodic β(1-6)-linked side chains. A similar polysaccharide can be found in grains, such as barley, which are common sources of dietary fiber.
The term "fiber" refers generally to a diverse, complex group of substances whose single common attribute is their resistance to human digestive enzymes. The important physiological effects of fiber are related to this characteristic, as well as to water and ion-binding capacity, viscosity, and the products of their fermentation in the bowel. E. Bright-See, Nutrition Today, 4:4-10 (1988). The implication is that fiber increases bulk, dilutes colonic contents, speeds transit time and changes bacterial metabolism, possibly providing protection from some diseases of the gut. Furthermore, diets high in fiber are usually lower in animal products, (i.e., saturated fat, cholesterol). By replacing or reducing these fats, protection against heart disease is enhanced. P. M. Kris-Etherton et al., Journal of the American Dietetics Association, 88:1373-1400 (1988); S. Bingham, American Journal of Clinical Nutrition, 45:1226-1231 (1987). Dietary fiber can alter lipid metabolism by interfering with cholesterol absorption, changing lipoprotein lipase activity or fatty acid metabolism. Also, fiber has been seen to lower blood glucose levels, alter hormonal levels and influence carbohydrate metabolism. Epidemiological evidence does suggest a link between reduced incidence of chronic heart disease and colon cancer in populations consuming high fiber diets. T. A. Miettinen, American Journal of Clinical Nutrition, 45:1226-1231 (1987); M. L. Wahlevist, American Journal of Clinical Nutrition, 45:1232-1236 (1987). Most authorities agree that dietary fiber intake should be increased from the current average of 10-20 gm/day to 25-30 gm/day, but not to exceed 45 gm/day. For most, this can be safely accomplished by consuming a variety of foods containing fiber.
It is known that a diet high in fiber is beneficial for a variety of reasons. Dietary fiber aids digestion because it provides an indigestible biomass which is carried through the alimentary canal causing undigested food to be pushed out before it. Fiber also clears out bacteria and insures the proper working of the peristaltic muscles. A diet high in fiber has been shown to reduce serum cholesterol and triglyceride levels, and has been linked to lower rates of intestinal and colon cancer. A high fiber diet has also been linked to the reduction of other problems commonly associated with poor digestion, such as ulcer formation, ileitis and colitis.
Dietary fiber is particularly important for persons on a liquid diet, such as, for example, a patient who receives nourishment through a feeding tube due to inadequate function of the gastro-intestinal tract, with or without protein calorie malnutrition. A common side effect for those patients who do not receive any fiber supplement is chronic diarrhea due to lack of bulk. This condition may cause depletion of essential nutrients in the patient, dehydration and bowel irritation. Some dietary fibers can be used as a stool or fecal bulking agent to aid digestion and control chronic diarrhea. A fiber having a large water holding capacity is useful for this purpose, because these fibers absorb excess fluid in the colon resulting in increased fecal wet weight and stool number. Furthermore, some fibers are partially digested by bacteria in the large bowel, forming short chain fatty acids, which are preferred energy fuels for the small intestine and improve intestinal function.
Grain β-glucans are commonly used as a source of fiber. Another source of β-glucans is yeast cell walls; however, yeast β-glucans have not been used as a dietary fiber supplement due to the fact that yeast cell wall glucans prepared by traditional methods, such as the method described by Manners et al. , in Biochem. Journal, 135:19-30 (1973), are not pure, and generally contain materials such as protein, glycogen and chitin. Also, the water-holding capacity of β-glucans prepared previously is low due to the presence of hydrophobic components such as protein and glycogen, and due to the lack of any three-dimensional cell wall micro-structure. Functionality and purity of a compound to be used for non-irritating, food-grade, fiber preparations are essential to ensure acceptable performance and qualitative properties, such as bland taste and white coloration.
The present invention is based on the discovery that whole β-glucans are surprisingly useful as dietary additives. Dietary supplements containing whole β-glucans, when administered to animals and humans, provide a source of fiber in the diet, provide a fecal bulking agent, provide a source of short chain fatty acids and reduce cholesterol levels in animals and humans.
In addition, this invention includes a method for providing a source of fiber in the diet, for reducing body weight, providing a fecal bulking agent, for providing a source of short chain fatty acids and/or for lowering total plasma and/or low-density lipoprotein (LDL) cholesterol by administering whole yeast β-glucan to an individual. Whole glucans are significantly more effective in reducing body weight or weight gain in animals when compared to commercially available fibers such as oat bran or wheat bran. The whole glucans used in this composition and method can be naturally-occurring or unmodified β-glucans, or can be chemically or enzymatically modified, by altering the β(1-6) and β(1-3) linkages. For example, partial acid hydrolysis or digestion with enzymes can be used to modify the β(1-6) or β(1-3) linkages, respectively. Such modifications are useful to control the water holding capacity of the whole glucan, thereby regulating the degree of gastroenteral bulking.
More importantly, the relative proportions of β(1-6) and β(1-3) linkages in whole β-glucan can be altered by genetic modification of the yeast from which it is derived. Mutant cells which exhibit changes in the structure of the yeast cell wall can be isolated by evaluating their resistance to digestion by hydrolytic enzymes. The changes in the structure of the whole β-glucans may be reflected in the shape of the extracted whole cell walls, in the water-holding characteristics or in the extent of bacterial conversion to short chain fatty acids. Whole β-glucans extracted from mutant cells can then be used as dietary additives for purposes commensurate with their properties.
The dietary additives of the present invention are based on the three-dimensional matrix structure of whole glucan, which is unique to whole glucan, and cannot be mimicked by other naturally occurring polysaccharides, including other β-glucan preparations. The composition and method of this invention utilize whole yeast glucan as a dietary additive to improve digestion or treat digestive disorders. For example, the composition can be used to: 1) provide a source of dietary fiber; 2) provide a stool or fecal bulking agent; and 3) provide a source of short chain fatty acids via bacterial fermentation in the large bowel, which improves digestion and benefits intestinal and colonic endothelial cells. The present composition and method also effectively lowers total plasma and/or LDL cholesterol, and reduces body weight or inhibits weight gain.
The whole β-glucans of the invention can be administered neat, in a biologically acceptable carrier or as part of a complete nutritional food.
Whole β-glucans have important properties that make them ideally suited for use as dietary supplements. For example, they have a high water-holding capacity, they are non-gelling, the water holding capacity can be easily adjusted, and they are natural and highly pure. Whole β-glucans provide a number of coincidental, beneficial effects. They provide, in combination, a source of fiber, a fecal bulking agent, a source of short chain fatty acids to improve digestion and stimulate mucosal proliferation in the intestine and a cholesterol-lowering effect. Whole glucans provide a uniquely concentrated source of dietary fiber which can be used to control weight gain when incorporated into a high-fat or hypercaloric diet. Whole β-glucans are obtained from yeast cell walls using a purification process which does not disrupt the three-dimensional cell wall matrix in the process of extracting the non-glucan components, thus enhancing their functionality as a water-absorbing and bulking agent.
FIG. 1 shows a plot illustrating the viscosity profiles of whole β-glucans obtained from Saccharomyces cerevisiae A364A, 374 and 377.
FIG. 2 shows a plot illustrating the viscosity profiles of whole β-glucan obtained from Saccharomyces cerevisiae A364A and 374 before and after treatment with laminarinase.
FIG. 3 shows a plot illustrating the viscosity profiles of whole β-glucans obtained from Saccharomyces cerevisiae A364A and R4 including viscosity profiles of R4 after treatment with acetic acid and glucanase.
FIG. 4 is a graph illustrating the reduction in hamsters of total and LDL cholesterol obtained with whole glucan compared to wheat bran and oat bran.
"Whole β-glucans" are glucans which maintain the intact, three-dimensional in vivo morphology of the cells from which they are derived. Whole β-glucans (also referred to herein as "whole glucans") are obtained from the cell walls of glucan-containing organisms using a purification process which does not disrupt the integrity of the cell walls in the process of extracting the non-glucan components.
Yeast cell walls are composed predominantly of glucans, which are glucose polymers, having mainly β(1-3) linkages with a smaller component of β(1-6) linkages. Whole β-glucan is the alkali-insoluble portion which can be derived intact from yeast cell walls using the process described by S. Jamas et al. in U.S. Pat. No. 4,810,646, the teachings of which are incorporated herein by reference. This process yields intact particles of β-glucan, which maintain the spherical, elliptical or rod shaped configuration of the β-glucan as found in vivo, which is referred to as whole glucan. Whole glucan is comparable in size and shape to whole yeast cells, and is composed almost entirely of β-glucan.
These hollow, three-dimensional particles are conducive to a high water holding capacity, in that they become filled with water upon hydration. The viscosity of an aqueous solution of whole glucan is indicative of its water holding capacity. Viscosity profiles of β-glucans derived from three different strains of Saccharomyces cerevisiae are shown in FIG. 1.
β-glucans obtained using this process are also very pure. The occurrence of other cell wall components, including glycogen, protein and chitin, is minimized when this process is used. Glycogen, protein and chitin are undesirable as the presence of these components tend to reduce the water holding capacity and the effective dietary fiber content of β-glucans.
Highly pure, whole β-glucans having a very high water holding capacity and high dietary fiber content can be obtained from any yeast strain. For example, the following strains, and mutants or variants derived from them, will yield whole β-glucans: Saccharomyces cerevisiae, Saccharomyces delbrueckii, Saccharomyces rosei, Saccharomyces microellipsodes, Saccharomyces carlsbergensis, Schizosaccharomyces pombe, Kluyveromyces lactis, Kluyveromyces fragilis, Kluyveromyces polysporus, Candida albicans, Candida cloacae, Candida tropicalis, Candida utilis, Hansenula wingei, Hansenula arni, Hansenula henricii and Hansenula americana. This whole β-glucan retains the three-dimensional morphology of the yeast cell wall in vivo, since the Jamas et al. extraction process does not cause rupture of the yeast cell walls. The β-glucan product obtained from this process is typically about 96-99% pure; and, since the cell wall structure is intact, it also has a significantly higher water holding capacity than glucans extracted using traditional methods, which lack the intact three-dimensional structure.
Whole yeast β-glucans have several important properties that make them ideally suited to function as dietary supplements. Whole β-glucans are natural, very pure, exhibit a high water holding capacity and are non-gelling. They are wholly composed of glucose units bonded together by β(1-3) and β(1-6) glycosidic linkages, which cannot be de-graded by human digestive enzymes, thus making them suitable as a source of dietary fiber. Additionally, as whole β-glucans pass through the large bowel, they can be partially digested by endogenous colonic bacteria without significant loss of their water holding properties. This fermentation process gives rise to short chain fatty acids (predominantly acetate, propionate and butyrate) which are beneficial to mucosal cells lining the intestine and colon. Sakata, T. and Engelhardt, W. V., Comp. Biochem. Physiol., 74a:459-462 (1983).
Whole β-glucans are able to absorb more than ten times their weight in water, which is useful in oral and tube feeding formulations and digestive aids to act as a stool bulking agent, and, thus, as a preventative to diarrhea. For example, whole glucan has a water holding capacity of between about 3 to about 12 ml/gram of dry material, but does not exhibit gelling properties, thus allowing it to be incorporated into liquid formulations at significantly higher levels than possible with existing fibers such as soy polysaccharide, psyllium fiber and other plant or grain fibers. Therefore, a typical daily dose for humans of from about 5 to about 50 grams of whole β-glucan can absorb from about 15 to about 600 ml of water from the gastrointestinal tract depending on the desired function for the user. For example, the functional properties of the whole glucan can be varied between high fiber/low bulking to high fiber/high bulking functions.
The water holding capacity of whole glucan can be altered by genetic, chemical or enzymatic modification, as described by Jamas et al. in U.S. Pat. Nos. 4,810,646, 4,992,540, 5,082,936 and 5,028,703, by Jamas et al., in Biotechnology and Bio-engineering, 28:769-784 (1986); the teachings of all of which are incorporated herein by reference. For example, whole glucan can be treated with an acid, such as acetic acid, after its extraction from yeast to reduce the degree of β(1-6) crosslinking, thereby increasing the water holding capacity of the whole glucan. Likewise, treatment with hydrolytic enzymes, such as laminarinase or β(1-3) glucanases, reduces the degree of β(1-3) linkages of the whole glucan, resulting in a decrease in the viscosity profile (see FIG. 2) and thus, a decrease in the water holding capacity of whole glucan.
The properties of whole glucans can be altered by genetic modification of the yeast cells from which they are derived. A culture of yeast cells can be induced to mutate by applying a mutating agent, such as ultraviolet radiation, for example. Changes in the structure of the yeast cell wall induced by the mutation can then be evaluated for effect on the morphology and structure of the whole β-glucan extracted from the mutant yeast. These changes may be reflected in the shape of the extracted whole cell walls, the degree of cross-linking, in the viscosity profile (see FIG. 3) which relates directly to water-holding capacity, and/or in the extent of bacterial conversion to short chain fatty acids. β-glucans extracted from the mutant cells can then be used as dietary additives for purposes commensurate with their properties. For example, a mutant strain of yeast, Saccharomyces cerevisiae R4 (NRRL Y-15903, Agricultural Research Service Culture Collection, 1815 N. University Street, Peoria, Ill.), was created by exposing a culture of Saccharomyces cerevisiae A364A cells to UV light for 25 seconds, as described by S. Jamas et al. in U.S. Pat. Nos. 4,810,646, and 5,028,703 by S. Jamas et al. in Biotechnology and Bioengineering, 28:769-784 (1986). The R4 mutant shows increased β(1-6) linkages in the glucan structure, which affects its degree of digestibility, and the resultant yield of short chain fatty acids in the large intestine. Mutant yeast cells may be produced by other methods known in the art.
By treating the glucans with mild acids, such as acetic acid, or glucanase enzymes such as laminarinase, to alter the β(1-6) or β(1-3) linkages, respectively, or inducing or isolating mutant yeast strains having altered β-glucan structures, it is possible to control the hydrodynamic properties of whole glucan, and to control product viscosity precisely for a particular end usage. Therefore, the degree of bulking, for example, can be controlled by administering whole glucan modified to have the desired water holding capacity.
The multiple functionality of whole glucans as a source of short chain fatty acids and as a hydrophilic water absorbing agent makes them valuable as dietary additives acting as a fiber supplement and/or a stool bulking agent that enhances bowel function. Enzymatic modification of whole glucan can allow a range of digestability (from about 15 to about 70% metabolizable glucose) and water holding capacity (from about 3 to about 12 ml/g of dry material). These properties can be utilized individually or in combination depending on the specific application. In addition, the present whole glucans can be used to lower cholesterol levels in individuals with hypercholesterolemia. Whole glucans can be administered alone or in conduction with a lipid-lowering diet to lower total plasma and LDL cholesterol.
A dietary additive, or supplement, is defined for present purposes as a food material which is administered to or taken by an animal or human to induce a beneficial or desirable physical effect or to supply one or more components which are missing in the individuals' diet.
The present whole glucan preparations can be administered orally, or enterally. The form in which the glucan will be administered (e.g., powder, tablet, capsule, suspension, solution, emulsion) will depend on the patient and the particular treatment. The quantity of the composition to be administered will be determined on an individual basis, and will be based in part on consideration of the condition of the subject, the subject's overall health, and the severity of the disorder being treated. The amount of whole β-glucans administered to an individual to provide a source of fiber should be the amount necessary to provide all or part of the individual's fiber requirement to aid digestion by providing an indigestible biomass to clear undigested food and other debris from the gastro-intestinal tract; ensure the proper function of the gastrointestinal tract; prevent dehydration and/or bowel irritation in the individual; clear out bacteria and aid proper working of the peristaltic muscles; prevent diarrhea, which can occur when there is insufficient fiber in the diet; and/or to reduce serum cholesterol and triglyceride levels. The amount of whole β-glucans administered to an individual as a bulking agent should be the amount necessary to provide water-adsorbing bulk to prevent dehydration and bowel irritation in the individual; to aid digestion; and to increase fecal wet weight and stool number, and to thereby control chronic diarrhea caused by excess fluid in the digestive system. For example, dosage levels can be from about 5 to about 50 grams of whole β-glucan per day; and will generally range from about 30 to about 40 g of whole β-glucans per day to provide a source fiber in the diet and/or as a bulking agent. The amount of whole β-glucans administered to an individual as a source of short-chain fatty acids should be the amount necessary to aid digestion and improve intestinal function and stimulate proliferation of mucosal cells in the intestines. The amount of whole β-glucans administered to an individual to reduce serum cholesterol should be the amount necessary to cause a reduction in total plasma and LDL cholesterol. When added to a high-cholesterol and/or high-fat containing diet, about 5% to about 8% by weight (based on total weight of food ingested) of whole glucan was more effective than wheat bran or oat bran at reducing total plasma and LDL cholesterol levels. The amount of whole β-glucan administered to an individual to promote weight loss or reduce weight gain should be that amount necessary to aid digestion, increase fecal wet weight, speed transit time of undigested food and insure the proper working of peristaltic muscles. For example, when added to a high-fat diet, about 5 to about 8% by weight (based on total food intake) of whole β-glucan was more effective than wheat or oat bran in reducing body weight and weight gain.
The composition may be administered orally, in liquid or solid form, either at room temperature or chilled, or enterally through a feeding tube. Glucan as a source of fiber can be administered alone, in a biologically acceptable carrier (e.g., saline or water) with other ingredients such as vitamins and minerals, or as part of a complete-nutritional food. For example, whole glucan can be administered as a component in a high fiber liquid food for oral or enteral feeding, by continuous or intermittent drip into a feeding tube (e.g., nasogastric, nasoduodenal, jejunal). Liquid formulations generally contain about 5 g whole glucan per 8 fluid ounces.
The composition of the present invention can optionally include, in addition to whole β-glucan, other components, which will be determined primarily by the manner in which the composition is to be administered. For example, a composition to be administered orally in tablet or powder form can include, in addition to whole β-glucan, a filler (e.g., corn starch, sucrose, lactose), a binder (e.g., carboxymethyl cellulose, gum arabic, gelatin), an adjuvant, a flavoring agent, a coloring agent, and/or a coating material (e.g., wax or plasticizer) and/or other nutritional supplements. A composition to be administered in liquid form can include whole β-glucan and, optionally, an emulsifying agent, a diluent (e.g., water, sterile saline) and/or a coloring or flavoring agent, or combined in a complete feeding formula to be administered orally or by feeding tube into the digestive tract. A complete feeding formula can contain all nutritional requirements. For example, such a feeding formula for oral or enteral administration could contain whole glucan (as the source of dietary fiber), water, a source of carbohydrate (e.g., sucrose, hydrolyzed corn starch), an oil (e.g., corn or soybean oil), selected sources of vitamins (e.g., choline chloride, ascorbic acid, alpha-tocophenyl acetate, niacinamide, calcium pantothenate, thiamine, riboflavin, phylloquinone, cyanocobalamin, vitamin D3); selected sources of minerals (e.g., potassium citrate, magnesium chloride, calcium phosphate tribasic, sodium citrate, potassium chloride, zinc sulfate, ferrous sulfate, manganese sulfate, cupric sulfate); a source of protein (e.g., soy protein isolate, calcium caseinate), and lecithin.
For a complete nutritional liquid food, to be used as the sole source of nutrition, the dosage for an average adult can be between about 1500-2000 calories per day. A complete nutritional formulation can have the following caloric distribution:
______________________________________Liquid Nutrition Plus Fiber - Enteral FeedingComplete Nutritional FormulationContents Grams/Liter % of Total Calories______________________________________Protein 20-60 8-23Fat 1-100 1-90Carbohydrate 40-220 20-91Glucan 10-50 0-10Vitamins* RDA 0Minerals* RDA 0Water 800-950 0______________________________________ *Provide recommended dietary allowance of all essential vitamins and minerals
For most individuals the calorie distribution can be: about 14-15% protein, about 30-32% fat and about 53-56% carbohydrate.
The invention is illustrated further by the following examples, which are not to be taken as limiting in any way.
Composition of Whole β-glucans From Strains Saccharomyces Cerevisiae A364A and R4
Whole β-glucan was extracted from strains Saccharomyces cerevisiae A364A and R4 according to the process described by Jamas et al. in U.S. Pat. No. 4,810,646.
Protein and hexose content of whole glucans
Protein content of the whole β-glucans was determined chemically using the Bio-Rad assay (Bio-Rad, Richmond, Calif.). In this procedure, glucan samples were suspended in water to a concentration of 5 mg/ml, and lysozyme solutions in the range of 0.2 to 1.0 mg/ml were prepared as standards. Duplicate 0.1 ml aliquots of the samples and standards were placed in clean, dry test tubes and 5.0 ml of the diluted dye reagent was added. The solutions were vortexed, and after 5 minutes, the optical density (O. D.) was measured at 595 nm. A water blank was used as a negative control.
Total hexose was measured in duplicate 100-fold dilutions of the glucan suspensions. A standard curve was prepared using glucose solutions in the range of 10-100 μg/ml. Duplicate 1 ml aliquots of the samples were placed in clean, dry test tubes and 1 ml of 5% (v/v) phenol was added. Then, 5 ml of concentrated sulfuric acid (H2 SO4) was added to each tube, the mixture was vortexed, and incubated at 37� C. for 15-20 minutes. The optical density was measured at 488 nm with a water sample as the blank. The results are shown in Table 1.
TABLE 1______________________________________Protein Content Protein Hexose Hexose/ProteinPreparation mg/ml mg/ml Ratio % Protein______________________________________A364A 0.071 8.74 123:1 0.80R4 0.082 8.81 108:1 0.91______________________________________
Chitin and Glycogen Content
Fourier-Transform Infrared (FT-IR) spectroscopy was used to detect the presence of chitin and glycogen, and peak integration was used to determine their relative levels. The characteristic glycogen peaks, which appear at a wave number of 850, 930 and 760 cm-1, were not detected in the whole β-glucan preparations, but were clearly present in the glucans extracted using an alternate process described by Manners et al. in Biochem. J., 135:19-30 (1973) (hereinafter the "Manners' preparation"). In addition the characteristic chitin peak, which appears at a wave number of 1550 cm-1 was significantly stronger in the Manners' preparation. The results are shown in Table 2.
TABLE 2______________________________________CONTAMINANT LEVELS DETECTEDBY FT-IR SPECTOSCOPYContaminant Whole Glucan Manner's Prep.______________________________________Glycogen 1.0 9.4Chitin 1.0 2.9______________________________________
Manner's preparation contains an approximately 10-fold higher level of glycogen, and a 3-fold higher level of chitin over the whole glucan preparation. Table 3 illustrates the significant compositional differences resulting from these procedures:
TABLE 3______________________________________COMPARISON OF BETA-GLUCAN PREPARATIONSComponent Whole glucan Manner's Prep.Content mg % mg %______________________________________Glucan 1000 96.2 1000 51.8Protein 2 0.8 351 18.2Glycogen 31 2.9 579 30.0______________________________________
Based on 1000 mg samples of glucan, the whole glucan sample contained only 33 mg of contaminants, whereas the Manners' preparation contained 930 mg. The contaminant mass displaces its weight in water from the glucan particles, so that the contaminated glucan has a considerably lower water holding capacity.
Treatment of Whole Glucan With Acetic Acid
Five hundred mg samples of whole glucan from S. cerevisiae prepared as described in Example 1, from strains 374, 377 and R4, were suspended in 250 ml of 0.5 M acetic acid. The suspensions were continuously stirred at 90� C. for 3 hours. At the end of this extraction, the remaining insoluble glucan residue was recovered by batch centrifugation at 5000 rpm for 20 minutes. The glucan residue was washed once in 200 ml distilled water, once in 200 ml dehydrated ethanol and twice in 200 ml dehydrated ethyl ether. The resulting slurry was dried in air at 37� C. for 12 hours. The initial suspension in acetic acid and the supernatant were assayed for total carbohydrate content to determine the proportion of the extractable β(1-6) glucan component. The white glucan powder obtained after drying was resuspended in distilled water to determine its viscosity profile (see FIG. 3). The results are shown in Table 4. As indicated by the results, the mild acid treatment -�increases the water holding capacity of the whole glucans by approximately 20% and therefore will have a commensurate effect on their bulking properties.
TABLE 4______________________________________Viscosity Profiles of Glucans Beforeand After Acetic Acid TreatmentYeast Viscosity in Centipoise, 25� C.Strain 2% 2.5% 3% 3.5%______________________________________Saccharomycescerevisiae374 (before) 1.5 2.8 7.1 14.2374 (after) 1.6 4.7 58.3 1879.0377 (before) 1.6 3.4 16.0 75.0377 (after) 1.7 4.7 58.3 1878.0R4 (before) 1.8 2.3 3.1 4.5R4 (after) 2.6 4.6 12.4 106.3______________________________________
Treatment of Whole Glucan with Laminarinase
Four hundred mg samples of whole glucan from Saccharomyces cerervisiae prepared as described in Example 1, from strains A364A, 374 and 377, were suspended in 400 ml of distilled water. Laminarinase (endo β(1-3) glucanase; Sigma Chemical Co.) in a concentration of 0.25 mg/ml in phosphate buffer at pH 7.0 was added. The solutions were incubated at 37� C. for 4 hours. At the end of the incubation the solutions were held at 70� C. for 15 minutes to deactivate the enzyme. The remaining residue was recovered by centrifugation at 5000 rpm for 20 minutes. The resulting glucan residues were diluted into a range of concentrations in order to obtain viscosity measurements of the laminarinase degraded glucan samples. Since the enzyme cannot be effectively removed from solution, a control experiment was performed as above containing the enzyme only. The readings from the control were used to correct the solvent viscosity accounting for the contribution of the enzyme to the macroscopic viscosity of the suspension. The results are shown in Table 5.
TABLE 5______________________________________Viscosity Profile of Glucans Before and AfterTreatment with LaminarinaseYeast Viscosity in Centipoise, 25� C.Strain 1% 2% 3% 3.5%______________________________________SaccharomycesCerevisiaeA364A (before) 1.4 2.7 13.7 127.3A364A (after) 1.2 1.6 3.1 6.4374 (before) 1.6 4.0 47.4 2630.7374 (after) 1.3 2.3 9.3 62.9377 (before) 1.6 3.4 16.0 75.0377 (after) 1.4 2.8 18.1 367.3______________________________________
FIG. 2 shows the viscosity profile of glucan derived from strain A364A before and after a 4 hour laminarinase digest. Digestion of whole glucans with hydrolytic enzymes such as laminarinase will partially degrade their three-dimensional matrix structure thereby reducing their water-holding capacity. This procedure can be employed to produce β-glucans with high fiber/low bulking function.
Modification of Whole Glucan Cell Wall Structure Through Genetic Modification of the Producing Strain
The following procedure is employed to produce and isolate mutant yeast cells having altered cell wall structures: Saccharomyces cerevisiae A364A is used in this example, however, other yeast strains may be used.
Strain A364A was grown in 100 ml YPD to mid-log phase. The cells were washed in buffer and divided into aliquots of approximately 5 ml in sterile glass petri plates. A UV exposure of 25 sec (30% survival) was used as the mutagenesis procedure. The cells were then suspended in 5 ml YEPD (1% yeast extract, 2% bacto peptone, 2% glucose) and grown under subdued light to a concentration of 5�106 CFU/ml. The cells were harvested, and protoplasts were prepared using 0.25 mg/ml laminarinase for 30 minutes. The suspension was then diluted with water to lyse osmotically sensitive protoplasts. The surviving cells then were grown in 5 ml YPD to a density of approximately 5�106 CFU/ml. The laminarinase treatment was repeated two more times using 1.0 mg/ml enzyme for 15 minutes. The candidates which resistance to laminarinase digestion were then streaked on YEPD agar (1.5%) plates. Single colony isolates were tested for resistance to the enzyme compared to the resistance of the control strain, A364A. The mutant referred to as R4, is available from the Agricultural Research Service, Number NRRL Y-15903. The water holding capacity of whole β-glucan derived from Saccharomyces Cerevisiae strains A364A and R4 is shown in Table 6.
TABLE 6______________________________________The Water Holding Capacity of Genetically Modified(R-4) and Unmodified (A364A) Whole GlucansYeast Water-holding capacityStrain Treatment (ml/g dry material)______________________________________A364A none 9.2A364A acetic acid 9.0A364A laminarinase 5.7R4 none 8.9R4 acetic acid 10.3R4 laminarinase 7.1______________________________________
A Complete High-Fiber Liquid Food Containing Whole β-Glucan
A high fiber liquid food for oral or enteral feeding was formulated using whole glucan as the source of dietary fiber. The composition contained the following ingredients: distilled water, hydrolyzed corn starch, sucrose, sodium and calcium caseinates, corn oil, whole glucan, minerals (potassium citrate, magnesium chloride, calcium phosphate tribasic, sodium citrate, potassium chloride, zinc sulfate, ferrous sulfate, manganese sulfate, cupric sulfate), soy protein isolate, flavorings, soy lecithin and vitamins (choline chloride, ascorbic acid, alpha-tocophenyl acetate, niacinamide, calcium pantothenate, thiamine chloride hydrochloride, pyridoxine hydrochloride, riboflavin, vitamin A palmitate, folic acid, biotin, phylloquinone, cyano-cobalamin, vitamin D3).
The formulation was prepared by adding the following components to 4 fl. oz. of distilled water:
______________________________________Contents grams______________________________________Sodium caseinate 3.5Calcium caseinate 5.9Corn Oil 8.8Sucrose 7.0Whole B - glucan 5.0Hydrolyzed corn starch 26.3______________________________________
The following vitamins and minerals were added:
______________________________________Vitamin A 850 I.U.Vitamin D 68 I.U.Vitamin E 7.7 I.U.Vitamin K 12 mcgVitamin G 51 mgFolic Acid 102 mcgThiamine (Vitamin B1) 0.39 mgRiboflavin (Vitamin B2) 0.44 mgVitamin B6 0.51 mgVitamin B2 1.6 mcgNiacin 5.1 mgCholine 102 mgBiotin 77 mcgPantothenic Acid 2.55 mgSodium 200 mgPotassium 370 mgChloride 340 mgCalcium 170 mgPhosphorus 170 mgMagnesium 68 mgIodine 25.5 mcgManganese 0.85 mgCopper 0.34 mgZinc 3.83 mgIron 3.06 mg______________________________________
All components were mixed until dissolved or suspended in a homogeneous mixture, and enough additional water was added to bring the volume to 8 fl. oz.
A Bulk Laxative Containing Whole β-Glucan
A bulk laxative was formulated by adding 5.g of whole β-glucan to 8 oz. of cold water, and stirring until the β-glucan was suspended in the water. This formulation can then be administered orally to a patient, up to 3 times daily to provide a laxative effect.
Method to Control the Bulking Properties of Whole Glucans by Chemical Modification
The bulking properties of the whole glucans are a direct function of their hydrodynamic volume. However, degradation of the glucan matrix by hydrolytic enzymes present in the large intestine may significantly impair this property. It is therefore important to control the bulking properties and degradation of the glucan particles for a variety of applications.
Glucan particles were prepared according to the method of Jamas et al. (U.S. Pat. No. 4,810,646) from Saccharomyces cerevisiae 377. A portion of the glucan particles was further extracted by suspending 500 mg of the glucan in 250 ml of 0.5M acetic acid and stirring for 3 hours at 90� C. The whole glucan and the acetic extracted glucan were digested with a mixture of hydrolytic enzymes containing β(1-4), β(1-3) and β(1-6) glucanase activities to simulate the conditions that the glucan will be exposed to in the large intestine. The bulking properties of the glucan before and after digestion were measured as was the amount of glucose released and the percent undigested fiber (see Table 7).
TABLE 7______________________________________Properties of Whole Glucans Before And AfterDigestion Initial Water Final Water Holding Holding Digestable Capacity Capacity Glucose % UndigestedPRODUCT (ml/gram) (ml/gram) (mg/gram) Glucan______________________________________Untreated377 9.1 6.5 250 75377/acetic 10.3 4.1 600 40acid______________________________________
The two products, untreated 377 and 377 treated with acetic acid behave significantly differently under simulated conditions. The results show that the whole glucan, 377, will provide high bulking properties throughout its residence in the intestine and does not degrade significantly. The acetic acid-treated material is more susceptible to enzymatic hydrolysis, yielding a higher quantity of metabolizable glucose per gram of fiber (Table 8). In this case, the product will benefit patients with impaired bowel function in which the fiber will provide a low bulking component and considerable nutrition to the epithelial cells.
Method to Control Degradation of Whole Glucans By Modifying the Ratio of β(1-6) to β(1-3) Linkages
The whole glucans consist of β(1-3)-linked glucose backbone chains with periodic β(1-6) branching. The relative ratio of the β(1-6) to β(1-3) linkages in the glucan matrix determines its resistance to digestion by hydrolytic enzymes. The employment of mutant or genetically engineered strains with altered β(1-6) linkages result in whole glucans with a range of properties as dietary fiber.
To demonstrate this, whole glucans from Saccharomyces cerevisiae strain A364A, and the mutant strain R4, were digested with a mixture of hydrolytic enzymes to simulate the conditions in the intestine. The results, shown in Table 8, illustrate how the ratio of linkages in the whole glucan can be used to produce a high bulking/low digestible fiber or a low bulking/high digestible fiber.
TABLE 8______________________________________The Effect of Linkage Ratio in Whole Glucans ontheir Function as a Dietary Fiber Initial Water Final Water Holding Holding Digestable Capacity Capacity* GlucosePRODUCT (ml/gram) (ml/gram) (mg/gram)______________________________________A364A 9.2 5.0 550R4 8.7 7.1 225______________________________________ *After enzymatic digestion
The results above demonstrate how the A364A whole glucan is degraded considerably by the hydrolytic enzymes providing a high level of metabolizable glucose representing approximately 50% of the total fiber. The modified whole glucan of strain R4, which has an increased ratio of β(1-6) to β(1-3) linkages, is more resistant to the hydrolytic enzymes thus maintaining over 80% of its bulking properties and providing a lower quantity of metabolizable glucose.
The Effect of Dietary Whole Glucan on Serum Cholesterol Levels of Hypercholesterolemic Hamsters
Syrian hamsters (Mesocricetus auratus) were chosen for their similarity to humans in blood lipoprotein profile. Forty hamsters were placed on a control diet for seven weeks. Control feed consisted of a Purina Mills Rodent Laboratory Chow (#5001) based diet to which was added 0.2% by weight cholesterol and 10% coconut oil. At seven weeks, hamsters were separated into four groups having means of total cholesterol between 267 mg/dl and 279 mg/dl. One group remained on the control diet. The other groups received a five percent by weight addition of oat bran, wheat bran or whole glucan (prepared as described in Example 1) to the control diet for an additional six weeks.
Initial blood samples were drawn from each hamster after four weeks on control diet, after which time they were divided and immediately placed on the fiber-containing diets. At two consecutive intervals of 3-4 weeks all groups were bled again. All blood samples were obtained via the retro-orbital sinus and collected into heparinized tubes. Approximately 500 microliters of blood were collected from each hamster at each bleed.
Plasma was partitioned by low speed centrifugation. Plasma total cholesterol, high density lipoprotein cholesterol and triglycerides were measured by enzymatic assay on a Beckman 700 system, as previously described. T. O. Tiffany et al., Clinical Chemistry, 18:819 (1972); W. H. Karge et al., The American Journal of Medicine, 86:19-23 (1989).
Groups were tested for variance before being placed on fiber diets to ensure no initial statistical difference between mean values of total cholesterol. To evaluate the effect of each fiber, individual animals were used as their own control. An analysis of variance was performed to determine if there was an effect of type or level of fiber. A paired-t-test was applied across each group to examine the difference between means of the hamsters while on control feed and then on fiber treated feed. In each group changes in variables were tested for correlation by a Pearson correlation matrix. A means separation test (Newman/Kuels, Tukey) was then used to detect significant differences of effects on plasma lipoprotein cholesterol and body weight between groups and against a control average.
No significant differences in pre-treated values for plasma cholesterol were observed between each treatment group due to randomization. Whole glucan fed at 5% by weight resulted in reductions in total cholesterol, (42%), LDL cholesterol, (69%) and a significant increase in HDL cholesterol (16%). Plasma triglycerides were not significantly altered by the whole glucan at this level. The results are shown in Table 9 and in FIG. 4.
TABLE 9__________________________________________________________________________EFFECTS OF DIETARY FIBER (5%) ON PLASMA LIPIDS AND LIPOPROTEINCHOLESTEROL TC HDLC LDLC TGDIET N PRE TREATED PRE TREATED PRE TREATED PRE TREATED__________________________________________________________________________mg/dl5% WHEAT 9 272a 223 110 110 162 110 99 110 � � � � � � � � 61 37 34 23 64 39 39 445% OAT 6 291 304 93 114 198 190 135 94 � � � � � � � � 61 51 16 29 61 48 72 285% WHOLE 9 308 180b 98 114c 210 66d 114 97GLUCAN � � � � � � � � 108 34 16 15 96 29 27 48__________________________________________________________________________ a Values represent mean � standard deviation of each diet group. b Significant at P < .009 c significant at P < .009 d significant at P < .004 TC = total cholesterol HDLC = high density lipoprotein cholesterol LDLC = low density lipoprotein cholesterol TG = triglycerides
The Effect of Whole Glucan On Serum Cholesterol Levels of Hamsters
Twenty-four Syrian hamsters were fed a control diet consisting of Purina rodent chow (#5001) and after an initial plasma cholesterol determination (see Example 9), were divided into 8 groups and randomized so that initial pre-treatment plasma cholesterol levels were not significantly different from each other. Afterward, all animals were placed on a hypercholesterolemic chow-based diet containing 10% coconut oil plus 0.2% cholesterol, and either no added fiber (control group) or containing 5% oat bran or 5% whole glucan. After 4 weeks, animals were bled and plasma lipoprotein cholesterol and triglycerides were analyzed as described in Example 9.
The results, shown in Table 10, indicate that whole glucan reduced total cholesterol by 13% compared to only 6% with oat bran. LDL was reduced by 15% in the whole glucan group compared to 4% in the oat bran group.
TABLE 10______________________________________PLASMA LIPID PROFILES OF HAMSTERS FED AHIGH CHOLESTEROL DIET SUPPLEMENTEDWITH OAT FIBER OR WHOLE GLUCANTREATMENTADDED FIBER CONTROL OAT BRAN WHOLE GLUCAN(wt/wt) 0 5% 5%______________________________________TOTAL CHOL 238 � 47* 224 � 48 208 � 25(mg/dl)LDL CHOL 114 � 52 110 � 37 97 � 37(mg/dl)HDL CHOL 124 � 24 114 � 30 111 � 20(mg/dl)TRIGLYCERIDES 135 � 43 99 � 38 74 � 17(mg/dl)______________________________________ *Values represent Mean � Standard Deviation for 8 animals per group.
Administration of Whole Glucan to Hamsters on a Pre-Established High-Fat Diet
Thirty-three Syrian hamsters (Mesocricetus auratus) were placed on a high-fat diet (Purina Mills Rodent Laboratory Chow #5001 with 0.2% by weight cholesterol and 10% by weight coconut oil) for seven weeks. On the seventh week the hamsters were separated into four groups and the body weight of each hamster was recorded. Three of the groups then received 5% by weight of either wheat bran, oat bran or whole glucan (prepared as described in Example 1) in their high-fat diets. The fourth control group continued on the original diet with no additional fiber supplement. After six weeks the body weights of the hamsters were recorded. The results are shown in Table 11.
TABLE 11______________________________________EFFECT OF DIETARY FIBER ON BODY WEIGHT NUMBER OF BODY WEIGHTa (grams)DIET GROUP ANIMALS PRE TREATED______________________________________WHEAT BRAN 9 108 � 12 100 � 8bOAT BRAN 6 102 � 6 113 � 11cWHOLE GLUCAN 9 112 � 8 91 � 6dCONTROL 9 111 � 7 108 � 6______________________________________ a values represent mean � standard deviation b significant at p < .021 c significant at P < .025 d significant at P < .001
As shown in Table 11, whole glucan effected significantly higher weight loss compared to either oat bran or wheat bran.
Effect of Whole Glucan on Weight Gain of Rats
Sixty male Sprague-Dawley rats were separated into six groups with similar average body weights. Each group was assigned to one of six diets which differed by the source and/or proportion of fiber added to them: control (no fiber) cellulose (5%), oat bran (43%) and whole glucan at 1%, 4% and 8% by weight. All the diets were based on the AIN 76A diet as described in "Dietary Fiber and Health", JAMA, 262:542-546 (1989) and by Shinnich et al. in J. Nutr.. 118:144-151 (1988) and contained 1% by weight cholesterol and 0.2% by weight cholic acid. As oat bran contains 18.6% dietary fiber, the oat group diet contained 43% by weight oat bran so as to compare to the 8% whole glucan diet. The animals were maintained on their respective diets for 20 days. Initial and final body weights and daily food intake were monitored and are shown in Table 12.
TABLE 12__________________________________________________________________________THE EFFECT OF WHOLE GLUCAN ON 20 DAYWEIGHT GAIN IN RATS20 INITIAL FINAL FEED 20 DAYFIBER WEIGHT(g) WEIGHT(g) INTAKE(g) WEIGHT GAIN__________________________________________________________________________Fiber 263.0 � 9.55 344.5 � 21.4 16.25 � 1.64 81.5 � 15.3Cellulose(5%) 262.6 � 7.96 339.3 � 11.0 17.59 � 0.72 76.7 � 6.6Oat Bran(43%) 262.9 � 8.08 336.2 � 15.3 16.69 � 1.05 73.3 � 9.4Whole Glucan(1%) 262.7 � 8.33 336.6 � 18.0 16.10 � 1.46 73.9 � 11.5Whole Glucan(4%) 262.5 � 7.69 331.2 � 19.0 16.48 � 1.29 68.7 � 14.3Whole Glucan(8%) 263.5 � 10.62 318.7 � 11.8a 15.82 � 1.52 55.2 � 11.4a__________________________________________________________________________ a significant at p < 0.05 (Two tailed Dunnett Test)
As shown in Table 12, only whole glucan at 8% by weight of the diet resulted in a significant reduction in weight gain without requiring a statistically significant reduction in food intake. Surprisingly, the oat bran did not induce significant reduction in weight gain even at the 43% level.
Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed in the scope of the following claims.
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