WO2001003675A1

WO2001003675A1 - Extrusion of biomass to make tablets with high lipid levels

Info

Publication number: WO2001003675A1
Application number: PCT/US2000/018662
Authority: WO
Inventors: Claus Christian Becker
Original assignee: Martek Biosciences Corporation
Priority date: 1999-07-09
Filing date: 2000-07-07
Publication date: 2001-01-18
Also published as: AU5923200A

Abstract

This invention provides tablets, comprising microbial cells or cell fragments, and at least 10 % of a fatty acid containing material, such as docohexaenoic acid (DHA) or arachidonic acid (ARA). Generally the tablet has a dense, rather than porous structure, and is substantially free of liquid oil on the surface. This invention also provides a process for forming the above tablets comprising a) preparing a paste from dry microbial cells or cell fragments, preferably with fatty acids or fatty acid residues present in the paste as constituents of the microbial cells; b) extruding the paste to form tablets; and optionally c) drying the tablets to less than 20% moisture residue.

Description

Extrusion of Biomass to Make Tablets with High Lipid Levels

BACKGROUND

Field of the Invention This invention is directed to tablets that contain 10% (by weight) or more fatty acid-containing compounds with minimal addition of excipients, and methods for production of such tablets.

Review of Related Art

Extruders have been used to process a wide variety of materials, including food and animal feed. The use of extrusion is described, for example, in U.S. Patent No. 4,006,266, which discloses extruded pet food containing 5-15% fat, 20-60% cereal grain or starch and 8-12% moisture. U.S. Patent No. 5,141,755 discloses pet food made from a mixture containing 10-30% farinaceous material and 4-35% vegetable or dairy fat; the mixture is extruded hot (265°C) to form cubes which are then dried. U.S. Patent No. 5,871,802 discloses pellets of animal feed which are produced by extruding at low temperature (<140°F) a mixture of grains and highly concentrated carbohydrate solution; this process can form stable pellets containing 3-8% fat.

Extruded products containing microbial cells have also been described. U.S. Patent No. 4,041,181 discloses a process in which proteinaceous material is fermented to lower pH and the fermented material (including cells) is mixed with a coagulable binder, then extruded, cooled and cut into desired pieces.

U.S. Reissue Patent No. 29,130 discloses a process in which harvested cells are flocculated with anionic polyelectrolytes (1-50% by weight of cells), and the flocculated cells are extruded into desirable shapes and dried. The resultant particles serve as immobilized enzyme catalysts. U.S. Patent No. 5,130,242 discloses a process in which harvested cells are mixed with ground grain to lower the moisture content below 40% and then extruded and dried to produce a dry product containing omega-3 fatty acids. In contrast, U.S. Patent No. 5,904,943 discloses a process in which filamentous fungi are mixed with a gel-forming hydrocolloid which is gelled to form a firm mass (paste) containing at least 50% fungi, 0.2-5% hydrocolloid, 0- 20% water and 1-5% fat. The mass is minced to produce particles, but there is no suggestion to extrude the paste.

Extrusion is used to make a host of products from pasta to plastics, but not to make dietary supplements or pharmaceuticals. In particular, biomass- containing tablets that deliver nutritional oils have not been produced in this way.

SUMMARY OF THE INVENTION

This invention provides tablets, comprising microbial cells or cell fragments as the major constituent (i.e., at least 50%, preferably 70%, more preferably 90% by weight), where the tablet contains at least 10% fatty acid- containing material, and preferably at least 4% polyunsaturated fatty acids or fatty acid residues, and no more than 20% moisture. Typically, the tablets are suitable for consumption by swallowing, i.e., 50-2000 milligram particles having dimensions from a few millimeters to a centimeter or so for humans and proportionally for animals. Generally, the tablet has a structure which is dense, not porous, and is substantially free of liquid oil on the surface. In a preferred mode, the polyunsaturated fatty acids or fatty acid residues comprise docosahexaenoic acid (DHA) and/or arachidonic acid (ARA), more preferably, these two fatty acids constituted the majority of the unsaturated fatty acids and/or fatty acid residues. In one mode the polyunsaturated fatty acids or fatty acid residues are supplied as constituents of the microbial cells. In one embodiment, the tablet comprises a carbohydrate binding agent in an amount less than the oil content.

This invention also provides a process for forming particles comprising microbial cells or cell fragments as the major constituent, where the particles have at least 10% oil, and preferably at least 4% polyunsaturated fatty acids or fatty acid residues and no more than 20%) moisture. The process comprises the steps of: (a) preparing a paste from dry microbial cells or cell fragments, preferably where polyunsaturated fatty acids or fatty acid residues are present in the paste as constituents of the microbial cells; (b) extruding the paste to form tablets; and optionally (c) drying the tablets to less than 20% residual moisture. The process of this invention may be used to form biomass-containing tablets having a dense structure which is not porous, without the necessity of compacting the biomass in a tableting mold.

This invention provides a low cost process (relative to molding, tabletizing or incorporation into soft or hard gelatin capsules) for producing a DHA-, ARA- (or one or more other nutritional substances) rich food, supplement or pharmaceutical. It eliminates the need for extraction of the DHA and/or ARA from the cells. The incorporation of nutrients into a dry form (<20% H₂0) allows them to be supplied as part of a small mass, which is shaped by extrusion, molding or tabletizing. Modest use of excipients and processing aid materials means that little or no dilution of desired compounds occurs. This allows a high concentration of nutrients to be delivered or the use of smaller or fewer tablets to deliver a target dose.

DETAILED DESCRIPTION OF THE EMBODIMENTS

This invention provides a way to prepare a dry form of DHA or ARA which is minimally refined or worked-up relative to the natural origin. It is also an inexpensive way to make, for instance, a tablet containing microbially derived lipid material. According to one mode of this invention, fatty acid containing biomass (preferably DHA- or ARA-containing) is extruded to produce a tablet containing fatty acids or fatty acid containing compounds. The tablet of this invention is a dry formulation for man and/or animal consumption; preferably, the tablet is suitable for swallowing by humans. Typically tablets designed to be swallowed are 2 grams or less in mass, preferably 1 gram or less. The biomass tablet of this invention is substantially homogeneous (i.e., any heterogeneity is randomly distributed with regard to any linear direction, although there will be microscopic differences, e.g., between the inside and outside of a given cell). The tablets are typically dry (< 20% moisture, more preferably <12-15% moisture), and contain at least 50% biomass, i.e., material consisting of cells (intact or broken) of animal, fungal, bacterial or plant origin or the corresponding dried fermentation broth or components found in spent growth media. Preferably, the tablets contain biomass at levels much greater than 50%, e.g., 70%) biomass, 85% biomass or even 90% biomass. Tablets according to this invention will typically contain compounds having fatty acid residues, i.e., lipids such as triglyceride oils or phospholipids or even free fatty acids. Generally tablets according to this invention will contain at least 10% lipids, more preferably at least 20%) lipids or even greater than 35% lipids. In a preferred mode, the fatty acid residues include polyunsaturated fatty acids, and the tablets may provide a source of ARA and/or DHA for human nutritional supplementation.

As contemplated by this invention biomass includes material produced by living systems, including material of animal, vegetable or microbial origin, including animal or plant meals, etc.. Lipids in the tablets of this invention include fats and fat-derived materials, which are relatively insoluble in water and soluble in organic solvents, such as fatty acid esters (including triglycerides and phospholipids), fatty alcohols, sterols, waxes, etc. These lipids may be from any source, including especially edible oils such as vegetable oil or fish oil or microbially derived oils. Biomass suitable for processing according to this invention may be obtained by fermentation of oleaginous microbes. Suitable microbes and fermentation methods include those described in U.S. Patent Nos. 5,658,767 (ARA production) 5,407,957 (DHA production by Crypthecodinium cohnii) and 5,130,242 (oil production by Thaustochytrium), all of which are incorporated herein by reference.

Tablets according to this invention may be made from dried biomass by extruding a biomass paste and shaping the extruded material. Dried biomass is typically 20%> or less moisture, but may have up to 25-30% moisture. The water content of dried biomass of animal, vegetable or microbial origin is adjusted to between twenty and fifty percent (by weight) more preferably between 30 and 40%, to form a moist powder, a paste or a dough. Typically the biomass contains lipid material comprising at least one percent or more of arachidonic acid or docosahexaenoic acid or another essential fatty acid. Other additives may be included in the wetted mass, paste, such as vitamins, including, but not limited to, tocopherol, minerals, including, but not limited to, calcium and zinc, and herbal extracts, including, but not limited to, gingko biloba and St. John's Wort, animal or microbial extracts, or carotenoids, for example zeaxanthan or lutein, or other oils or lipids such as phospholipids (e.g. phophatidyl serine) or triglycerides. The mix is forced through a cylinder or a barrel by means of one or more rotating screws or pistons. Kneading and shearing action may be augmented by the addition of screens and breakers. The powder, paste or a dough is extruded through a die to form the desired two dimensional cross-section. At the end of the cylinder the material is forced through a die or a nozzle to provide the desired cross section. Then the extruded material is cut to the desired thickness to produce a tablet. The thickness of the product may be adjusted by the speed of knives which cut slices off the material as it exists through the die or nozzle. Typically, the thickness of the product will be adjusted to produce tablets of 0.5-2 gram size.

As biomass paste is extruded through the orifice of an insert in the die at the end of the cylinder, movement of the paste does not match the conditions known as "plug flow". Rather, the friction between the paste and the wall of the orifice retards flow of the paste near the wall, while paste farther from the wall comes out faster. When the exiting material is cut with a flat knife, the result is a tablet which is flat at the bottom and rounded at the top. By cutting with a curved blade that cuts deeper at the center of the extruded biomass string (that is, the knife is convex when observed from the insert, and symmetrical around the axis of the insert), the bottom of the tablet becomes rounded. Between cuts, more material will pass through the the center of the die insert than near the wall of the insert where it will be held back by friction. If the curvature of the knife is not too great relative to the speed differential between biomass paste at the wall and that at the center of the insert, the result is a rounded top. Having a rounded top and bottom facilitates swallowing of the tablet, and the tablet looks more esthetically pleasing. Tablets without flat edges are also much easier to coat than flat ones, which tend to get stuck together in a process known as "twinning". This happens when capillary forces bind together two flat sides of tablets after they are wetted with the coating material. Cutting to produce rounded ends would also be of benefit to products that are not intended for coating (e.g. pet food) as the more obtuse angles would make the product flow better and be less friable. The more obtuse the angle, the less it is susceptible to chipping. Chipping is not just a cosmetic problem; it can compromise the coating of a tablet and lead to loss of active material and/or premature release of the contents, or otherwise interfere with the functionality of the dosageform. The process of this invention typically uses dried biomass which requires the addition of a minimal amount of water for handling in the extruder and optionally a binding agent (e.g. starches, sugars or gums). However, an advantage of the process described herein is that high oil content biomass may be tableted without the addition of chemical binding agents, thereby providing a more natural material. The process also avoids the additional step of compacting the material in a tableting mold. For high oil biomass, it has been found that oil will bleed out of tablets made by conventional compacting of biomass containing 20-25% oil. Tablets made by the process of this invention are dry, dense particles with no visible surface oil and minimal oil bleed, and they are not subject to crumbling, i.e., they are not friable. Since nutrients, such as polyunsaturated fatty acid residues, are allowed to remain mostly inside the cells, the nutrients are protected from atmospheric oxygen, prooxidants such as iron, and other insults. Therefore, this is a gentler process than hard or soft gelatin encapsulation of extracted oil or biomass

Coating of the extruded particles/tablets may be used to make the tablets more aesthetically pleasing, less odiferous, and/or more stable (e.g., reducing the effect of environmental factors such as oxidation by reducing access by oxygen to the components of the tablet). This can be achieved, for example, by a coat of gum Arabic or similar. It may also be helpful to determine DHA levels in the tablets as they come out of the extruder to better control the process. Where the biomass is homogeneous, this problem may be reduced to the determination of biomass concentration. Due to the salt content of typical biomass, conductivity may be a quick way of measuring this.

In lab experiments the biomass from a microbial strain which has a higher content of DHA has demonstrated better properties during extrusion and drying. The higher DHA content also provides for a smaller tablet to contain the same amount of DHA. In addition, production costs for the extruded tablet will be significantly lower than the cost to make a soft-gel capsule containing extracted oil as a source of DHA (for equivalent DHA content).

This invention is an improvement over alternative processes for providing essential fatty acid supplements by extracting oils out of biomass, and either incorporation them into a dry form and tabletizing it, or the manufacture of soft-gel oil-containing capsules. Extrusion provides an all-natural product at minimal cost. Thus, this invention provides essential fatty acid supplements which are more natural and less expensive than the alternatives, and also may be prepared with no animal components. It is also a gentle way of manufacturing supplements containing polyunsaturated fatty acids, such as DHA and/or ARA. This technology might also be used to make mixed products (DHA + gingko biloba, for example). In another alternative, the process provides a low cost avenue to DHA products for other applications such as aquaculture. In particular, by controlling the particle size and/or the density of the extruded material (for example by steam expansion on exiting the extruder , as is known in the art), PUFA containing particulates for aquaculture feeds may be prepared.

EXAMPLES

In order to facilitate a more complete understanding of the invention, a number of Examples are provided below. However, the scope of the invention is not limited to specific embodiments disclosed in these Examples, which are for purposes of illustration only.

Example 1. Extrusion of biomass from Crypthecodinium cohnii

Spray-dried Crypthecodinium cohnii biomass from Martek Biosciences prepared as described in U.S. Patent No. 5,407,957 was extruded using a single screw extruder. 120 kg of biomass was put into the hopper and fed into the mixer under vacuum (23-25 inches). Water (40°C) was added to a final concentration of 36%. The result looked dry, but had a crumbly, wet sand feel to it. The color was ocher. From the mixer, the mixture went into the extruder. Feed rate was 250 kg per hour. Transit time was 10-15 minutes. Upon exit, a rotary knife cut the output into rice-grain-sized particles. The water content was 36%. The color was dark brown. The texture was firm dough.

The product was conveyed into a continuous forced air drier where it was dried at approximately 45°C for seven hours. After five minutes the moisture level was 34%. After 65 minutes the water content of the tablets was 26%. After three hours of drying the value was 15%. After seven hours it was 7.8%.

Example 2. Extrusion of biomass from Crypthecodinium cohnii

Extrusion was carried out as above, except icing sugar from a local market and sorbitol from SDI Polyols, Inc., was added to the feed to see if it would act as a binder. Five bags each containing 20 kg of biomass, one kg of icing sugar and 100 grams of sorbitol were added to the hopper. Appearance and drying were as above. Following extrusion and drying, the tablets appeared lighter in color and smoother in appearance than after the same operations in Example 1. Example 3. Extrusion of biomass from Crypthecodinium cohnii

Extrusion was carried out as in Example 1 , but with maltose from SDI Polyols added to see if it would act as a binder. Five bags each containing 20 kg of biomass and two kg of maltose were added to the hopper. The color of the extruded product was darker and the surface was smoother. After drying the differences were less pronounced.

Example 4. Extrusion of biomass from Crypthecodinium cohnii

This experiment tested the use of tablet sized inserts in the extruder. Successful tableting was not necessarily expected, since large tablets (>0.5 cm thickness) made with durum wheat will crack during drying. Tablet-shaped dies were ordered from Niccolai Dies USA, St. Louis, MO.

Crypthecodinium cohnii biomass was used in which the concentrated biomass had been washed to reduce NaCl levels prior to spray dying.

Four new die inserts were mounted in a circular die at the same distance from the center, since this position gives the straightest extrusion. Biomass was extruded and cut into tablets by a rotary knife, then dried. Some of the tablets that came out of the extruder were somewhat bent out of shape. This was a result of the impact they were subjected to when expelled from the extruder. Tablets dried very well. No cracking observed. This may be due to the oil content. The approximate weight of dried tablets was 1 gram.

Example 5. Extrusion of biomass from Crypthecodinium cohnii

Extrusion was carried out as described in Example 4. 67 kg of biomass with 1% rosemary extract and 15 mg/kg of Ascorbyl Palmitate was placed were placed into the hopper. Rosemary extract was Herbalox dry HTD 20, supplied by Kalsec, Inc. Ascorbyl palmitate was the standard AP. Flow from hopper into extruder was a problem because of bridging. Vacuum couldn't be maintained and went up to thirteen inches. Tablets that came out had deep ridges on the sides but smooth top and bottom. Addition of more water (up to 31% of total) improved the smoothness. After drying the tablets looked good, with no cracking, and they were not friable.

Example 6. Extrusion of biomass from Crypthecodinium cohnii

Extrusion was carried out as described in Example 4. 40 kg of biomass with 7% semolina wheat flower. Semolina flower was granular semolina from Amber Milling, St. Paul, MN. As expected, the addition of wheat flower improved shape and smoothness of tablets. Moisture content was 36%. After drying the tablets looked good, with no cracking, and they were not friable.

Example 7. Extrusion of biomass from Crypthecodinium cohnii

This experiment tested whether extrusion of biomass would work with six smaller tablet size inserts. These were designed to make smaller tablets to reduce the effect of impact after extrusion, and the inserts were Teflon coated to give smoother sides. The flow problem experienced during the previous round was solved by agglomeration of biomass.

Teflon coating provided a clear improvement: sides of tablets were smoother than the top and the bottom, and the top of the tablets was rounded while the bottom was flat. There was no problem with flow of biomass into the mixer. Water going in was approximately 37°C. Vacuum was 26 inches. The approximate weight of the dried tablets was 500 mg.

Example 8. Extrusion of biomass from Crypthecodinium cohnii

Extrusion was carried out as described in Example 7. Biomass (90%) was mixed with Durum wheat flour (5%) and Maltose (5%). Maltose was

ADVANTOSE 100 (SPI Polyols, Inc.). Water content was increased during the experiment from about 25% to about 35%. The best product came out when the motor was consuming 11 amps. When at 10 amps the product looked too wet and at 12 amps it looked too dry (although all product made dried very well). Example 9. Extrusion of biomass from Crypthecodinium cohnii

Extrusion was carried out as described in Example 7. Biomass (95%) and Gum Arabic (5%) was mixed. Gum Arabic was Instant C from Colloides Naturels. Water content was about 35%). The mix looked grainy. After extrusion it resembled the other products. Gum Arabic didn't seen to give better binding or improve firmness of tablet.

Example 10. Coating of Biomass Tablets

Tablets from example 4 and Example 7 were coated with hydroxypropylmethyl cellulose (80% Surelease/20% Opadry II, mixed and sprayed on tablets at 10% w/w in a side-vented pan-coater, Labcoat II, O'Hara Technologies). The coating appeared uniform and appeared to reduce odor. No oil bleed-through was observed after coating. (No significant bleed through was observed for any of the particles of Examples 1-9 before coating.)

Claims

1. A tablet comprising microbial cells or cell fragments as the major constituent, said tablet having at least 10% fatty acids or fatty acid-containing compounds.

2. The tablet of claim 1, wherein the tablet is no more than 20% moisture.

3. The tablet of claim 1, wherein the fatty acids or fatty acid containing- compounds comprise docosahexaenoic acid (DHA) and/or arachidonic acid (ARA).

4. The tablet of claim 1 , wherein the microbial cells contain fatty acids or fatty acid-containing compounds.

5. The tablet of claim 1, wherein the tablet comprises a carbohydrate binding agent in an amount less than the oil content.

6. The tablet of claim 1, wherein the tablet is substantially homogeneous.

7. The tablet of claim 6, wherein the homogeneous tablet is surrounded by a coating.

8. A process for forming particles comprising microbial cells or cell fragments as the major constituent, said particles having at least 10% fatty acids or fatty acid residues and no more than 20% moisture, said process comprising the steps of: preparing a paste from dry microbial cells or cell fragments comprising fatty acids or fatty acid residues as constituents of the microbial cells; extruding said paste to form tablets; and drying the tablets to less than 20% residual moisture.

9. The process of claim 6, wherein the tablet is at least 90% microbial biomass.