Field of the Invention

The present invention relates to edible compositions containing structured

triglycerides produced from genetically engineered non-tropical annual crops.

Background of the Invention

Most of the body of knowledge that has been built up over the years regarding

the functionality of lipids in food systems has been based on experiments designed

around variations of naturally occurring fats and oils. The functional performance of

these fats, from whatever source, was related to specific analytical characteristics that still enjoy wide use in the industry, i.e., Solid Fat Index, Iodine Value, and Fatty Acid

Composition. In addition to these characteristic values, a number of analytical tests

were routinely performed on the fats that were indicators of its quality, or its ability to

withstand the stresses of temperature and shelf-life requirements. These included free

fatty acid content, peroxide value, color, odor, etc. None of these tests, however,

related^ the functional performance of the fat to the presence (or absence) of any specific

triglyceride having a specific structure, i.e. , which fatty acid was on which carbon of

the glycerol backbone. In the majority of cases, however, such knowledge would have

been of academic interest only since the availability of such structured fats in any great

proportion within a given fat system were simply not available. Aside from cocoa

butter and some of the other more exotic tropical fats, most fats used in foods consist

of a random assortment of triglycerides driven by the types and levels of fatty acids that

make up their composition, so that such knowledge would have no direct bearing on

a formulator' s capabilities.

Cocoa butter makes up approximately one third of chocolate's content, and it

is responsible for the much-appreciated characteristics of chocolate. Such

characteristics include chocolate's hardness and brittleness at ambient temperatures,

quick and complete melting in the mouth, gloss, shelf life, aroma and taste.

It is believed that the carbon chain length of the fatty acids making up cocoa

butter as well as the symmetry of the acids positioned on the glycerol moiety are

responsible for the much-appreciated characteristics. In fact, three fatty acids

completely dominate the composition of cocoa butter: palmitic acid, stearic acid and

oleic acid. Practically all oleic acids occur esterified in position two of glycerol, with

two fully saturated fatty acids, stearic and palmitic acids, occupying the two remaining

positions — positions one and three. As reported in Cocoa Butter Alternatives,

Karlshamns Oils & Fats Academy, page 9, ^® 1991 (herein incorporated by reference),

this gives rise to three completely dominant symmetrical triglycerides, POP (palmitate,

oleate, palmitate), POST (palmitate, oleate, stearate) and STOST (stearate, oleate,

stearate), that resemble each other closely and make up almost 80% of cocoa butter.

Because of this symmetry, cocoa butter is deemed a symmetrical triglyceride.

However, because cocoa butter is expensive, and its supply is limited, various

researchers have spent considerable amounts of time in developing fats that could serve

as alternatives to cocoa butter by providing similar properties to its melting profile and solids content at various temperatures. Several classes of alternatives have been

developed, ranging from cocoa butter "equivalents" produced from selected blends of

fractions of natural fats that are high in specific triglyceride contents, and which are

miscible in all proportions to cocoa butter, to the use of fats containing totally different

triglyceride distributions, but which mimic, in many ways, the melting behavior of

cocoa butter. An example of the production of a cocoa butter "equivalent" would

involve the purification and frationation of a series of different naturally occurring fats

to obtain the proper proportion of the desired triglycerides having the desired structure, at the appropriate levels. Commonly used sources of these specialty fats and their

triglyceride distributions are given in the table below. The majority of the POP portion

required is obtained from palm mid-fraction.

Recently, a great deal more effort has been expended against the study of

structured triglycerides in foods, and these have led to the market introduction of

synthesized species that have been almost exclusively targeted at the confectionery

market for the replacement of cocoa butter, with the additional benefit of producing

reduced calorie products. These products take advantage of the effects of positional

isomerism on the glycerin backbone to address the specific physical properties required

in the final food product, plus utilize the differences in caloric contribution of the

various fatty acids used to arrive at a lowered calorie intake. The drawback of these

novel ingredients is that they are costly to manufacture, and require a series of synthetic

steps along with requisite purification procedures. The final price to the end-user is

still several dollars per pound. At this price, their ultimate use is restricted to specific

niche markets within the food industry rather than being able to effect a significant

move towards their use in a wide array of food products.

The assignee of the present application has developed genetically engineered

annual plants that will selectively produce laurate canola oil. These plants are

described in U.S. Patent 5,344,771, herein incorporated by reference.

In laurate canola oil, two fully saturated fatty acids of equal length occupy

positions one and three of the glycerol moiety in a majority of instances, and a C18

fatty acid occupies position two of the glycerol moiety in substantially all instances.

The symmetry in the engineered triglyceride is not as prevalent as found in cocoa butter, and for this reason, the triglycerides of the invention are deemed structured

triglycerides. In essence, the preferred and dominant occurring structured laurate

canola oil looks like this:

C - C - C

C12:0 C18:X C12:0

wherein X in the C18 fatty acid is 0, 1, 2 and 3, or the C18 fatty acid may be partially

hydrogenated. Hydrogenation will allow for variation in the Solid Fat Index profiles

of resulting fats.

The assignee has also produced a structured stearate oil, wherein, again, fully

saturated fatty acids of a carbon length of eighteen carbon atoms occupy positions one

and three of the glycerol moiety a majority of the time and a C18 fatty acid is found at

position two substantially all of the time which may be or may not be partially hydrogenated. Such a structured lipid is described in International Patent Application

No. PCT/US91/01746, herein incoφorated by reference.

The assignee has now found that substitution of the structured lipids for

conventional shortening compositions in food products enhances a host of

characteristics of such products.

Summary of the Invention

A first embodiment of the present invention relates to the manufacture of various food products having enhanced properties such as greater whiteness or enhanced flavor release by replacing conventional non-structured or natural triglyceride

lipids traditionally used in such food products with genetically engineered structured

lipids.

A second embodiment of the invention is directed to reduced fat or lower fat compositions using the genetically engineered triglycerides of the invention.

A third embodiment of the present invention relates to the use of the structured triglycerides in combination with conventional emulsifiers. Stable oil-in-water emulsions are produced using such systems and it is found that the use of structured lipids allows for a reduction in the amount of emulsifier required to create an oil-in- water emulsion, than is required using non-structured lipids.

A fourth embodiment of the invention is directed to the use of the structured

lipids of the invention as replacements for cocoa butter in certain chocolate flavored

coatings.

These and other objects of the invention will become apparent by reference to

the detailed description of the present invention.

Brief Description of the Drawings Fig. 1 is a comparison plot of transmittance evidencing whiteness.

Fig. 2 is a comparison plot of bread compression resistance over time intervals.

Fig. 3 is a comparison plot of cake compression resistance.

Fig. 4 shows effects of hydration on Solid Fat Index curves of final fats.

Fig. 5 shows the effects of hydration on a particular structured laurate canola oil. Detailed Description Of The Invention

The following terms used in the present disclosure are defined as follows:

Engineered structured lipid or triglyceride - any triglyceride produced by a

genetically engineered annual plant wherein a majority of the fatty acids at positions

one and three of the glycerol moiety are similar. /. e. , of equal carbon length and

substantially fully saturated, and the fatty acid at position two is substantially a C18:X

fatty acid wherein X is 0, 1 , 2 and 3 or the C18 fatty acid may be partially hydrogenated.

Non-engineered or natural lipids - any triglyceride produced by non-genetically

engineered plants such as soya bean, rape and turnip rape, cotton, coconut palm, cocoa,

illipe, shea, etc. Although cocoa and other tropically grown trees may produce

symmetrical lipids, these lipids are not produced by annual plants, and are thus not included in the definition immediately above.

β' form - a crystal form of fatty acids showing two peaks of variable intensity at 3.9 and 4.2 Angstrom.

CNO - Coconut oil

IV - Iodine Value, a measure of degree of unsaturated fat. Lower values indicate greater saturation.

PKO - Palm kernel oil

SFI - Solid Fat Index, a measurement related to a percent of solid fats found at a specified temperamre. A plot of specific volume versus temperamre is used to calculate the percentage of solid fat at any given temperamre.

Shortening - an edible fat or triglyceride which may contain one or more additives.

The manufacture of food products using engineered structured lipids generally requires no special techniques. One merely substitutes the engineered shortening for conventional shortening of the art. What is special and surprising is that based on hedonic scoring, reflectances measurements, and other objective and subjective tests, applicants have unexpectedly found that food products containing the engineered structured lipids have, inter alia, enhanced flavor release, good mouth feel, excellent spread properties, and superior whitening ability. The food compositions of the invention will include a genetically engineered structured lipid or triglyceride.

These compounds include triglycerides having the formula c — C — C

I I I

R ^l :0 C18:X R ² :0

wherein R ¹ and R ² , at least for a majority of the fatty acids at these positions, have carbon lengths of twelve or greater, are substantially completely samrated and are of

equal chain length. Preferably R ¹ and R ² are lauric and stearic acid, but may include the fatty acids of myristic and palmitic or in general fatty acids of C 12 or greater.

Generally, R ¹ and R ² comprise 66 mole % of the fatty acids of the triglycerides

and C18:X comprises 33 mole % of the fatty acids. In practice, for instance, up to

40% by weight and preferably up to 58% by weight or greater of R ¹ and R ² in laurate

canola oils are samrated C12 and C14 fatty acids (substantially C12), and for a majority

of the oil produced by structuredly engineered plants. R ¹ and R ² are found to make up about 36-38 weight % of the fatty acids of the triglyceride. See International

Application Number PCT/US95/03997 herein incorporated by reference. The amounts

of strucmral laurate oils ultimately obtained from the seeds of the plants may reach

99% .

A C18:X fatty acid is found at position two substantially exclusive of other fatty

acids; C18:X represents a fatty acid of eighteen carbon atoms so that X=0, 1, 2 and

3, or C18:X is partially hydrogenated. Thus, the fatty acid found at position two may

be a mixture of stearic, oleic, linoleic and linolenic fatty acids, and, if not partially

hydrogenated, it will be predominantly unsaturated.

Selective hydrogenation of this oil, then, allows one to closely control the

melting properties of the resulting triglyceride, and to manipulate the solids profile of the final fat system. What is noted when these new fats are utilized in typical

formulated food systems is that the flavor release offered by these systems was far

superior to those offered by the corresponding tropical laurics.

In general, food compositions of the invention will include, in addition to the

genetically engineered triglyceride, one or more of the following: salts, sweeteners,

water, protein sources, starches, other oils and fats, emulsifying amounts of oil-in-

water emulsifiers, food dyes, vitamins, antioxidants. gums, artificial flavor and the

like, as well as combinations thereof. Although the present invention is not limited in its broadest aspect to specific ingredients or ranges, generally the sweetener will be present in amounts up to 10-70% by dry weight of the formulation. The sweetener may be any of those conventionally used in the production of food products. Preferably, a

substantial portion of the sweetener is dry corn syrup solids. Other suitable sweeteners include maltodextrin, sucrose, fructose, dextrose, etc.

The protein will comprise 4-25% of the formulation. Such proteins include, for instance, soy protein, non-fat milk solids, whey solids, fish protein, a water-soluble or

dispersible salt of calcium such as calcium caseinate or sodium casemate, or a cottonseed protein, yeast protein, etc. Flours include wheat such as semolina or other wheat flour, rice flour, legume flour, oat flour, rye flour, corn flour, etc. Flour may be present in the amounts of 15-50 % .

The genetically engineered lipid is present in amounts of 1-99% . The above percentages do not preclude the presence of minor ingredients in the formulation such as flavoring agents, buffers, and/or stabilizers. Minor amounts of an antioxidant such as butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), tertiary butyl hydroquinone (TBHQ), citric acid, propyl gallate, and tocopherols such as vitamin E

may be used. TBHQ is preferred. Emulsifiers which can be used with the triglycerides of the invention include

crystallizable emulsifiers such as an alkali or alkaline earth metal salt of an acyl lactylate, such as sodium and calcium stearyl-2-lactylate. Any normally hard or soft

mono-and diglyceride having a monoglyceride content from about 30 to about 90% can

be used.

Selection of an oil/water emulsifier for the shortening of the present invention

is within the skill of the art. In this regard, the emulsifier can be any emulsifier used

in the preparation of oil/water emulsions. The emulsifier should be fat soluble or dispersible when used at the level necessary for the emulsification desired.

Any normally hard or soft mono-and diglyceride having a monoglyceride

content from about 30 to about 90, the remainder being mainly di-and tri-glycerides,

and an IV of about 4 to about 85 can be employed. See. for instance, U.S. Pat. No.

4,239,786, herein incoφorated by reference.

The amount of the emulsifier employed should be sufficient to maintain an

oil/water emulsion of, for instance, a whitener formulation. For this puφose,

preferably a plastic emulsifier is employed. At functional levels, hard emulsifiers could

cause set-up or excessive viscosity of the fluid shortening formulation. With an

emulsifier such as a mono- and diglyceride, for a spray dried product, about 9-10%

based on the weight of the shortening maintains the whitener formulation as an

emulsion form until spray drying takes place.

Another suitable emulsifier is Dur-Em 114 (trademark, SCM Coφoration), a

mono- and diglyceride derived from soybean oil having a monoglyceride content of

about 40% , and IV of 70-75 and a Capillary Melting Point of about 110°-120°F.

For a liquid whitener, it is preferable to employ a blend of emulsifiers, such as

EC- 117, (trademark, SCM Coφoration) a blend of mono- and diglycerides and lactylic

esters of fatty acids. Such emulsifier has a WICLA (Water Insoluble Combined Lactic

Acid) of about 4.8% (miriimum), a mono-diglyceride content of about 24-32% , and a

Capillary Melting Point of about 123°-133°F. This emulsifier provides freeze-thaw

stability for the liquid whitener. For a liquid whitener suitable for refrigeration

storage, where freeze-thaw stability is not necessary, a mono-diglyceride in the form

of a white bead from hydrogenated vegetable oils (containing citric acid) having a

mono-diglyceride content of about 40% (minimum), an IV of about 5 max. , and a

Capillary Melting Point of about 145° - 150° F, can be used.

Other fat soluble or dispersible emulsifiers which can be used in the shortenings

and in coffee whiteners of the present invention include distilled monoglycerides; ethoxylated fatty acid esters such as ethoxylated mono- and diglycerides; acyl lactylates

such as sodium or calcium stearoyl-2-lactylate; succinylated mono- and diglycerides;

propylene glycol monoesters; and polyoxyethylene-containing fatty acid esters such as

polysorbate 60, sorbitan esters and ethoxylated sorbitan esters.

For a spray dried product, blends of emulsifiers which can be used include, for

instance, a mono- and diglyceride with a lactylic ester of a fatty acid.

The ethoxylated fatty acid esters, and their manufacture, are described in Egan

U.S. Pat. No. 3,433,645, incoφorated herein by reference. The fatty acid radicals are

higher fatty acid chains preferably having about 12 to about 18 carbon atoms.

Ethoxylated mono- and diglycerides are the polyethoxylated fatty acid esters of glycerol

and may conveniently be described as mixmres of stearate, palmitate and lesser amounts of myristate partial esters of glycerin condensed with about 18 to 22 moles of

ethylene oxide per mold of α-monoglyceride. Santelle EOM is manufactured from

hydrogenated vegetable oils and has an acid value maximum of 2, an hydroxyl value

of 60-80, and an IV number based on fatty acid content of 3 maximum and an

oxyethylene content of 60.5 - 65.0% .

Useful polyoxyethylene-containing fatty acid esters are the polysorbates such

as polyoxyethylene sorbitol distearate, polyoxyethylene sorbitan monostearate,

polyoxyethylene sorbitan monolaurate, as well as other similar ethoxylated fatty acid

esters. An example of a suitable propylene glycol monoester is Myverol P-06 (trademark, Eastman Chemical) having an IV of about 5 and a congeal point of about

113.

Blending of the structured triglyceride and the disclosed emulsifiers can be

achieved in a monomer as disclosed in U.S. Pat. No. 4,239,786, herein incoφorated

by reference. What is remarkable is the fact that the emulsifiers can be used in reduced

amounts with the structured triglycerides as compared to amounts needed to maintain

oil-in-water emulsions with the namral triglycerides or non-engineered triglycerides.

Of course, the compositions will ultimately be consumed via a food product. For

whiteners, the product may be a beverage, for example, one produced from the leaves, seeds, pods, beans, bark, fruit or roots of a plant, such as coffee or tea, etc.

The following examples illustrate the present invention.

A conventional coffee whitener formulation has the following composition:

Vegetable fat 10.00%

Corn syrup solids (35 D.E.) 10.00%

Sodium caseinate (Erie 400) 1.25%

Emulsifier (DUR-Em44) 0.2-1.0%

Buffer 0.1-0.5%

Stabilizer 0.02-0.5%

Other 0.4%

Water Balance

About one-half of the solids of a typical coffee whitener formulation is vegetable fat,

wherein the fat may include partially hydrogenated vegetable oils such as palm kernel,

soybean and cottonseed. One such vegetable oil product is sold by Van Den Dergh as

Paramount X.

Examples

All the examples below disclosing specific triglycerides should be used as

guides and not as absolutes, as many strucmred triglycerides composed of different

ratios of fatty acids may be used.

Whitening of, for instance, as related to a coffee whitener, occurs as a result of

light being reflected from the surface of the finely divided fat globules within the

product. The engineered strucmred lipid or triglycerides used in compositions of the

invention produce crystals that enhance the whitening properties of the product. This

enhanced whitening allows for the production of reduced fat formulations.

Liquid whiteners of the invention are prepared by co-melting the engineered

strucmred triglyceride of the invention with an emulsifier and adding this melted

composition to dipotassium phosphate (buffer) in cold water.

A dry mix of corn syrup solids, sodium caseinate. lactylate and carrageenan are

added to the aqueous mixture with additional water, if necessary, and with vigorous

mixing to dissolve all solids. The mixmre is heated to 120°F and remaining ingredients

according to the conventional formulation are added. The mixmre is pasteurized at

160°F for 30 minutes, homogenized at 2500 + 500 psi and cooled rapidly to 40°F.

The composition is to remain refrigerated between use. The following fat and reduced

fat formulations were produced.

Example 1 Example 2

Full Fat Reduced Fat

Formulation. % Formulation. %

Water 77.35 80.35

Engineered Strucmred Lipids 10.00 7.00

Corn Syrup Solids, 35 DE 10.00 10.00

Sodium Caseinate ¹ 1.25 1.25

Emulsifier 0.50 0.50

Dipotassium phosphate 0.30 0.30

Carrageenan ³ 0.20 0.20

Sodium Stearoyl Lactylate ⁴ 0.40 0.40

Sources:

1. Erie Foods. Erie 400

2. Van Den Bergh, Dur-Em 114

3. FMC, Viscarin GP 109

4. American Ingredients Co.

The engineered strucmred triglyceride of the full fat formulation used as an

example of a strucmred triglyceride in Example 1 is a laurate canola oil; it has a melting point of 95 °F an IV of 45, and is further characterized as:

Fattv Acid Weight %

C8:0 0.0

C10:0 0.1

C12:0 34.8

C14:0 3.8

C16:0 3.0

C18:0 5.5

C18: l 45.8

C18:2 3.3

C18:3 0.8

C20:0 0.6

C22:0 0.6

C24:0 0.1

Other 1.6

Solids Profile

Temp °F % Solids

50 34.5

70 15.5

80 1.6

92 0.3

100 0.1

104 0.1

Saturates Profile

Total Saturates 49.0

Total Monosaturates 46.9

Total Polyunsaturates 4.1

Of course, this example using a specific triglyceride is for puφoses of illustration and explanation and should not be inteφreted as limiting the invention in any way. Preferably, the C12:0 and C14:0 fatty acids of the strucmred triglycerides

used in the present invention are present in the triglycerides in amounts of between 30-

40 weight percent and up to 59% or more. These weight percents are obtained from

the genetically engineered plants producing the strucmred triglycerides.

The engineered strucmred triglyceride of the reduced fat formulation (Example

2) is a laurate canola oil; it has an IV of 35, a melting point of 100° F, and is further

characterized as follows:

Fattv Acid Weight %

C8:0 0.0

C10:0 0.1

C12:0 35.3

C14:0 3.5

C16:0 3.2

C18:0 18.7

C18: l 37.1

C18:2 0.2

C18:3 0.3

C20:0 0.8

C22:0 0.6

C24:0 0.1

Other 0.1

Solids Profile

Temp °F % Solids

50 59.7

70 49.6

80 38.8

92 20.7

100 0.0

104 0.0

Saturates Profile

Total Saturates 49.0

Total Monosaturates 46.9 Total Polyunsaturates 4.1

Again, this is a non-limiting example.

Fig. 1 is a comparison of percentage transmittance of blends of 7-10 % laurate

canola oil as the vegetable oil component in a coffee whitener, as compared to

"Paramount X," a partially hydrogenated palm kernel oil with lecithin recommended

by Van Den Bergh. As shown, 9 and 10% blends of laurate canola oil exhibited less

transmittance, i.e. , greater reflectance, than Paramount X (the control) and thus imparts

greater whiteness. Also as shown, reduced fat compositions containing laurate canola

oils perform similarly to the Paramount, especially at the higher wavelengths.

Example 3

Crackers can be prepared from the partially hydrogenated vegetable oils of the

invention having excellent snap, improved flavor release over crackers containing

conventional shortenings, desirable tender mouth feel, and with minimal bleeding of fats to the surface of the cracker. These crackers can be produced, for example, with

the ingredients as shown in the table below:

Batch

Ingredient Weight. % wt„ grams

Sponge

Flour, moisture 8.8% (1) 50.73 650.80

Genetically Engineered Lipid 10.13 130.00

Distilled water @ 100°F 16.91 216.90

Yeast 0.17 2.20

Malt Syrup 0.78 10.00

Dough

Flour, moisture 8.8% 16.76 215.00

Salt 0.67 8.60

Baking Soda/Sodium Bicarbonate 0.34 4.30

Calcium Phosphate 0.16 2.10

Distilled Water 3.35 43.00

The sponge is prepared by heating the water to 100°F and dissolving yeast in

the water. The engineered triglyceride used in Example 1 is heated to 150°F. The

engineered triglyceride and the heated water with yeast are combined, malt syrup and

flour are added. The ingredients are then blended and molded into a cohesive mass. The sponge is placed in a bowl, covered, and set in a proofer at 80°F for eighteen

hours. Thereafter, the sponge is removed and mixed with dough ingredients of Table

I for four minutes, and then the mixmre is placed in a covered bowl in a proofer for

four hours. The dough is sheeted to one millimeter and folded several times to obtain

sixteen layers. The sheeted dough is docked on both sides, cut to fit an aluminum

T U 97/ 6 37

20 baking sheet, and then baked at 500°F in a convection oven for 4.5 minutes. The

crackers are cooled and then sealed in a plastic bag. As discussed above, these

crackers had excellent snap, improved flavor release as compared to crackers

containing conventional shortenings, desirable tender mouth feel, and minimal fat bleeding to the surface of the crackers.

Examples 4 and 5

Icings and reduced fat icings based on the engineered strucmred triglycerides of the invention can be produced. Suggested formulations for producing icings are set

forth below.

Example 4 Example 5

Standard Reduced-fat

Formula, wt.% Formula, wt.%

Ingredients

Sugar, 6X 70.10 70.10

Genetically Engineered Oil 19.50 —

Genetically Engineered Oil — 14.50

Water 5.80 10.80

Mono-and diglycerides ^m 2.00 2.00

Corn Syrup, 42 DE 1.95 1.95

Vanilla, 2X 0.40 0.40

Salt 0.25 0.25

¹ Van Den Bergh, Dur-lo™

The icings are prepared by combining the engineered strucmred triglyceride

with the emulsifier from Van Den Bergh and heating the same until both are well melted. Thereafter, salt and sugar are dry blended, and corn syrup and the fat mixmre

are added. The ingredients are mixed for three to four minutes at slow speed. Water

and flavoring such as vanilla can be added, and the composition is mixed until smooth.

Example 4 was prepared with the genetically engineered triglyceride of

Example 1 and the reduced fat formula of Example 5 was produced with the genetically

engineered triglyceride of Example 2.

Both the standard formula and the reduced- fat formula exhibited excellent

spread properties, excellent mouth feel, and superior flavor release. It is also noted

that the genetically engineered triglycerides provide the formulator with the oppormnity to produce reduced-fat versions without sacrificing product quality.

Example 6

Vegetable dairy cream cheese containing genetically engineered triglycerides.

A vegetable dairy cheese product is obtained by combining the following ingredients :

Ingredients Weight %

Water 62.00

Genetically Engineered 32.00

Lipid

Sodium caseinate ⁽¹⁾ 4.50

Locust bean gum ^(2> 0.50

Salt, NaCl 0.50

Emulsifier ⁽³⁾ 0.50

Flavor t.s.

Sources:

(1) Erie Foods, Erie 400

(2) Hercules, FL-50-50

(3) Van Den Bergh, Dur-Em 114

The cheese is prepared by dry mixing sodium caseinate, locust bean gum, and salt. This dry mix is added to the genetically engineered triglyceride of Example 2 maintained at 160° F in amounts as set forth in the table above. This mixmre was added to water which was preheated to 160°F. The mixmre was homogenized at 2,000 +

500 psi. The homogenized mix was heated to 170°F and an emulsifier was added. The

mixmre was pasteurized at 170° F for thirty minutes. Thereafter, antioxidants such as anhydrous citric acid and glucono delta lactone were added and the composition was again homogenized at 2000 + 500 p.s.i. A vegetable-based cream cheese was packaged hot and stored at 40°F. The cheese exhibited excellent mouth feel and superior flavor release. The pH of the vegetable oil-based cream cheese should be 4.2-

4.3 after 24 hours storage at 40 °F.

Cream fillings and reduced fat cream fillings can be prepared with the triglyceride of the present invention. The following full fat and reduced fat

formulations were prepared.

Example 7 Example 8

Full-Fat, 25% Reduced-Fat

Ingredients Formulation. Vft, % Formulation, wt. %

Standard Filling Fat 25.60

Genetically Engineered Oil — 2.50

Emulsifier ⁽¹⁾ 2.50 2.50

Sugar, 6X 45.90 45.90

Milk Solids 6.00 6.00

Salt 0.20 0.20

70°F Water, 1st Addn. 3.70 5.70

70°F, 2nd Addn. 9.30 11.30

70°F, 3rd Addn 6.80 9.20

Flavor t.s. t.s.

Sources: Van Den Bergh, Dur-lo ^τ

In order to prepare the cream fillings, the dry ingredients were blended at a low speed for one minute. The genetically engineered strucmred triglyceride or shortening was added to the blend at low speed for one minute. The first addition of water was added and the ingredients were mixed at a medium speed for five minutes. The bowl

was then scraped. A second addition of water was added at a medium speed for five minutes and again the bowl was scraped. A third water mix was added at medium

speed for five minutes.

The cream fillings had improved flavor release and mouth feel. As shown, the

cream fillings can be produced by reducing the amount of triglyceride 25 % . The

triglyceride used in both Examples 7 and 8 was the triglyceride of Example 1.

In another suφrising embodiment of the invention, it is found that the

genetically engineered triglycerides of the invention can be combined with cocoa butter.

What is particularly suφrising is that it can be used when cocoa butter makes up

greater than three percent of chocolate-flavored confectionery coatings. In fact, it is

known in the art that all lauric fats differ from cocoa butter and other non-iauric oils

and fats in composition. Because of this difference in composition, cocoa butter and

lauric fats are incompatible. In view of this incompatibility, lauric fats could only be

used, prior to the present invention, as substimtes for cocoa butter. In practice, cocoa

butter substitutes, such as the high lauric acids, can only be used together with low-fat

cocoa powder (10% to 12% fat content) as the miscibility of these two fats is poor. On

the other hand, cocoa butter replacers such as oils rich in C16/C18 are very miscible with cocoa butter. This compatibility allows a chocolate manufacturer to use cocoa

powder with higher fat content or a chocolate flavored liquor in their formulas, creating

confections with excellent chocolate flavor. No such flavor advantage is found with

the lauric fats. However, it has been found, suφrisingly, by the present inventors,

that the C 12 genetically engineered strucmred triglycerides of the invention can be used

as cocoa butter replacers as they exhibit good miscibility with cocoa butter. In fact,

cocoa butter can be used in amounts of greater than 3 % , up to 6 % , and possibly in even

greater amounts, with the strucmred laurate triglycerides of the invention while imparting an excellent chocolate flavor to confections.

The following formulations represent recommended starting formulations for use in chocolate-flavored confections for enrobing or molding applications. Variations in the types and levels of cocoa butters used will affect the final flavors of the finished

product. Two different types of genetically engineered strucmred C12 triglycerides are used in the examples below. Blending ratios of these triglycerides may significantly affect the melting and texmre profiles of finished products . One of ordinary skill in the art will be able to adjust the ratio based on melting points and the SFI of the blends to

those closely matching processing and organoleptic needs of a particular product.

Example 9

A milk chocolate-type compound coating will contain the following ingredients.

Ingredient Weight u&

Sugar, 6X 49.60

Cocoa Powder, Namral (10-12) 11.00

Non-fat Milk Powder 8.00

Genetically Engineered Lipid 28.00

Genetically Engineered Lipid 3.00

Lecithin 0.30

Vanillin 0.10

Processing includes dry blending ingredients such as sugar and cocoa powder, and non-fat milk powder. Thereafter, lecithin is added to an engineered strucmred lipid

and melted. Sufficient milk is added to the mixed powders to form a refinable paste. The paste is refined in a three roll mill to approximately 20 microns. The mixmre is

conched overnight and then molded into bars. The engineered lipid present in larger

amounts is a laurate canola oil having a melting point of 104° and an IV of 25. It is

further characterized as follows:

Fatty Acid Weight Saturates Profile

C8:0 0.0 Total Saturates % 69.7

C10:0 0.1 Total Monounsaturates % 30.0

C12:0 34.9 Total Polyunsaturates % 0.3

C14:0 3.5 Total 100.0

C16:0 3.3

C18:0 26.2

C18: l 30.0

C18:2 0.1

C18:3 0.1

C20:0 1.0

C22:0 0.6

C24:0 0.1

Other 0.1

Total 100.0

Solids Profile

Temperamre °F % Solids

50 66.2

70 59.2

80 52.0

92 30.6

100 2.2

104 0.0

Again, the fatty acid composition of this example and those to follow are

intended for illustration and do not limit the percent weight of the fatty acids in any

way.

The second triglyceride, i.e. , the one present in amounts of three percent by

weight, has an IV of 15 and is further characterized by the following:

Fatty Acid Wt. % Saturates Profile

Total Saturates % 84.5

C8:0 0 0..00 Total Monounsaturates % 15.0

C10:0 0 0..11 Total Polvunsarurates % QΔ

C12:0 3 366..00 Total 100.0

C14:0 4 4..00

C16:0 1 1..55

C18:0 4 411..55

C18: l 1 122..55

C18:2 0 0..11

C18:3 0 0..22

C20:0 1 1..22

C22:0 0 0..11

C24:0 0 0..11

Other 2 2,,77

Total 100.0

Solids Profile

Temperature °F % Solids

50 75.7

70 75.2

80 73.8

92 67.8

100 45.0

104 21.3

Most lauric fats crystallize in the beta prime form, without the need to go

through any elaborate tempering step during the processing of the coating mass.

Through X-ray crystallography , laurate canola oil was found to crystallize into a beta-

prime crystal, with no polymoφhism exhibited. With this knowledge, the product of

Example 9 was evaluated in a standard confectionery coating formulation vs.

commercial lauric fats based on both PKO and CNO. The results of these experimental

evaluations revealed some significant diffrences between the laurate canola coatings and

those made using PKO or CNO-based fat systems, including:

•Significant increased flavor impact with laurate canola.

•Increased coating shelf life (decreased bloom) when using laurate canola.

• Preferred mouthfeel over standard laurics.

Typical lauric fats have limited compatibility with cocoa butter, and tend to

produce eutectic effects in admixmres that create softer fats than either of the two base

fats alone. This is primarily caused by the interference of the crystallization path of the

lauric fat on the crystallization dynamics of the cocoa butter. The addition of only a

few percent of cocoa butter into a typical lauric fat will result in this softening effect,

causing the resulting fat blend to be unsuitable for use in coating applications. With laurate canola, however, such negative interactions with cocoa butter do not occur until

significant levels of cocoa butter (ca. 40% on an oil basis) were admixed. This means

that sources of "chocolate" flavor, typically those high in cocoa butter, can now be used

to impart more of the desired flavor to the finished goods when laurate canola is used

as the base fat for the coating. The acmal mechanism of this co-crystallization effect

has not yet been determined, but the functional effects of such blends, as inteφreted

through SFI curves of the laurate canola/cocoa butter systems, is clear.

In general, one can combine different combinations of laurate canola oils, i.e.,

oils of different hydrogenation.

Fig. 4 shows that the slopes of Solid Fat Index curves become steeper as the

level of C12 increases. It is also apparent that the tailing of the curves toward the

higher melting end (the so-called waxy portion of the curve) is minimized as the C 12 content increases.

Fig. 5 shows that, in examining a specific laurate canola having approximately

38% C12 content with various degrees of hydrogenation, i.e., by varying the content

of C 18:0 vs. C18, unsamrates that occupy the sn-2 position the following effects on the SFI (Solid Fat Index) curve are noted.

As a reference, the SFI for palm kernal stearine is also provided. The solids

profile that it provides is bracketed by two laurate canola products; one with an IV of

25 and one with an IV of 35. With this data, one can effectively match the solids

profile, at least in the melting range around body temperamre, that most manufacturers require for their products.

Example 10

Milk chocolate-type coatings can be prepared for enrobing using the following

ingredients.

Ingredient Weight, %

Sugar, 6X 49.60

Cocoa Powder, Namral (10-12) 11.00

Non-fat Milk Powder 8.00

Genetically Engineered Lipid 31.00

Lecithin 0.10

Vanillin 0.10

Procedures for making such coatings are the same as above. Products can be

molded into bars for subsequent use, or used directly for application on baked products.

These products have excellent flavor release, good compatibility with cocoa

butter, they do not require tempering, they exhibit excellent shelf life and superior

texmre (mouth feel and melt down). The products also exhibit excellent snap and

gloss.

The engineered triglyceride of Example 10 is the triglyceride used in minor

amounts in Example 9.

Example 11

In an attempt to better quantify the flavor release properties of strucmred lipids

of the invention versus other conventional oils when used in model food systems,

testing of flavor perception using a standard butter flavor was suspended in the

strucmral lipids of the invention at equal levels. Following are the results of these

tests:

METHOD USED:

1. Unflavored popcorn was air popped.

2. Each test oil was melted and a controlled amount of butter flavor admixed.

3. Popped popcorn was weighed into 50g portions.

4. Melted oil/flavor mixmre was sprayed over the popcorn while it was being agitated.

5. The popcorn and oil blend was weighed after oil application to assure that the

oil was on the popcorn, and did not include the amount on the walls of the

tumbling vessel.

6. Flavored popcorn was tested using a blind taste panel of five experienced tasters .

FORMULAS AND MATERIALS USED:

Ingredient Source Weight %

Popcorn Orville Reddenbacher 89.13 Fine Salt Morton's Popcorn Salt 00.36 Butter Flavor Tastemaker 330568 00.32 Oil Variable 10.20

Variables: Test 1 a: strucmred lipid IV 45 b: Code 321 (VDB)

Test 2: a: strucmred lipid IV 45 b: strucmred lipid IV 35 c: Satina 34 (VDB) d: Code 321 (VDB)

RESULTS

Test 1 : The lipid IV 45 was strongly preferred vs. Code 321 (a high stability

spray oil). 4 of the 5 panelists preferred the flavor from the IV 45, while the 5th

panelist had no preference.

An additional flavor test was conducted to isolate flavor release properties from

the individual fats - at least on an organoleptic basis — from those effects that might be

caused by differences in solids at body temperamre.

Test 2: Test 1 was repeated with the additional inclusion of two additional fats:

IV 35 (SFI @ 92°F = 10.7%) and Satina 34 (VDB, SFI @ 92°F = 1.0%). Previous

fats included were IV 45 (SFI @ 92°F = 0.3%) and Code 321 (SFI @ 92°F = 3% -

8%). The scoring of Test 2 is shown below, with the lowest numbers representing the

most preferred fat base in terms of flavor release (hedonic scoring was 1 = like

intensely; 4 = dislike intensely). Acmal scores given represent total sums from all panelists.

Fat Score SFI <a> 92F

Satina 34 6 1.0 strucmred lipid 10 0.3

IV 45

Code 321 16 3-8 strucmred lipid 18 10.7

IV 35

The results of this test confirm (directionally) the preference for the flavor

release provided by strucmred lipid IV 45 vs. Code 321 , a fat currently marketed as a spray-on for flavor carrying. There is no directional indication, however, that SFI at

92 °F is a determinant or participant in the flavor release process.

In order to provide more objective data regarding flavor release, tests were

conducted to determine the release rate of three aroma compounds, ethyl acetate, ethyl

caprolate and limonene, in three model food systems: a pure oil system, an oil-in-water

emulsion, and a water-in-oil emulsion, wherein the oil was either strucmred laurate

canola oil or a random laurate canola oil.

Release rates were determined using a flame ionization detector in a GC system.

The pure strucmred oil (Laurical) appears to delay or slow the release of ethyl

acetate greatly as compared to random lauric oil. Strucmred or random oil type had

less effect on aroma release for the ethyl hexanoate and limonene. It appears that ethyl

hexanoate was released more slowly from the random oil between 1 and about 3

minutes, but had a very similar release profile compared with strucmred oil. either

before or after that period. Limonene was generally released more slowly from the

randomized oil. The data from these experiments was very reproducible.

Differences in release were noted for the oil-in-water and water-in-oil systems,

but were not as dramatic as those encountered for the pure oil systems.

The remaining examples are directed to food compositions containing

engineered structured oils or triglycerides prepared from strucmred triglycerides other

than lauric canola oil.

Generally, breads and cakes emulsified with strucmred stearate oil showed

softer crumb at the end of test periods than did controls produced with conventional

oils. Breads with unemulsified engineered strucmred stearate oil and unemulsified conventional canola oil performed nearly identically. The emulsified genetically

engineered stearate oils had a softer crumb throughout the tests. The oil and emulsifier

levels used throughout resulted in a very narrow crumb texmre range, resulting in a

more uniform and consumer-acceptable product.

Example 12

BREADS

Breads can be prepared from the oils of the invention. These breads can be produced as follows:

FORMULAS

Ingredient Batch Percent. % Weight, g

Sponge

Flour, Bread 175.1 35.10

Water, Distilled 123.7 24.79

Yeast 5.0 1.00

Yeast Food 1.2 0.24

Dough

Flour, Bread 116.7 23.39

Water, Distilled 30.9 6.19

Sugar, Baker 19.9 3.99

Nonfat Dry Milk 5.0 0.98

Salt 5.0 1.00

Calcium Pro 0.3 0.06

Oil, Variable 16.2 3.25

Total: 498.9 100.00

Oil Variables

All oils were used at the same level.

1. Van den Bergh Food's Beta Plus™

2. Canola oil, Wesson retail product with sodium stearyl lactylate (Oil 95 % , SSL

5%).

3. Canola oil, Wesson with emulsifiers (Oil 79.6%, Van den Bergh's Tally 100™ 15.4%, SSL 5%).

4. Calgene Stearate Canola Oil (Oil 95 % , SSL 5 % ) .

5. Calgene Oil with emulsifiers (Oil 79.6%, Tally 100™ 15.4%, SSL 5%).

Emulsifiers were mixed in the oils and heated to 170°F, mixed until homogeneous and cooled to room temperamre prior to use.

The breads were prepared by blending together the sponge ingredients except

for the yeast. Water was heated to 100°F and yeast was dissolved in the water. Water

and yeast thereafter were added to the dry ingredients. The ingredients were mixed

until a cohesive mass was obtained. The mass was covered with plastic wrap, placed in an insulated box, and allowed to ferment for four hours. After the four hour

fermentation, the sponge was removed. It was a puffy, spidery, sticky mass, and was

kneaded briefly to fit into a Farinograph. The sponge, dough ingredients, and water

were placed into the Farinograph. The cover was closed and it was started for 30 seconds and stopped while the sides were scraped down. Thereafter, mixing continued

for an additional 6.5 minutes. The dough was removed, cut into balls of 225 gram

portions. The portions were rolled, turned and folded three times. On the final roll,

the roll was stretched into a pup pan sized cylinder where air was excluded as much as

possible from the pan. The dough was then placed into a pan and the pan was warmed

to 85° F in a proofing cabinet. The dough was allowed to rise 1.5 inches over the pan

(for about 90 minutes). A convection oven was preheated to 360°F and the dough was

baked for seventeen minutes. The bread was removed from the pan. The bread was allowed to cool and was sealed in plastic bags. One inch bread slices were tested for

compression resistance on days 1, 3, 5 and 7, with a TA-XT2 texmre analyzer. The

results are shown in Fig. 2.

RESULTS/DATA

Compression

Variable Resistance in Grams

Day 1 Day 3 Day 5 Day 7 Van den Bergh Beta Plus 253 433 634 656 Canola Oil, Wesson retail 319 519 900 871 Canola Oil, emulsified 256 480 722 769

Calgene High Stearate Oil 294 501 917 867 Calgene Oil, Emulsified 146 324 470 550

Discussion

Unemulsified Calgene laurate canola oil and conventional canola oils performed nearly identically in the breads over the life of the test.

Beta Plus™ control and emulsified Wesson canola breads had nearly the

same softness at day 1 , but the Beta Plus™ bread was significantly

softer by day 7.

Breads made with the emulsified Calgene laurate canloa oil started softer

and remained softer throughout the test period.

There is some variability in the hand forming method used in this test that can result in inconsistencies that may impact results (i.e. , the

incoφoration of air spaces, areas of slightly denser crumb strucmre, and

"firm" ridges at roll seams). We tried to minimize these variables as

much as possible.

38

Example 13

CAKES

Seven series of cakes were prepared with the following ingredients:

FORMULAS

Series 1,2,3,4,5 Series 6 Series 7

Ingredient grami _%_ grams %. grams Ά

Cake Flour 288.0 22.40 288.0 22.57 288.0 22.75

Sugar, Bakers 330.0 25.66 330.0 25.87 330.0 26.07

Nonfat Dry Milk 36.0 2.80 36.0 2.82 36.0 2.84

Salt 9.0 0.70 9.0 0.71 9.0 0.71

Baking Powder 18.8 1.46 18.8 1.47 18.8 1.49

Egg White, Dry 27.0 2.10 27.0 2.12 27.0 2.13

Water, Distilled 477.0 37.10 477.0 37.39 477.0 37.68

Fat, Variable 100.0 7.78 90.0 7.05 80.0 6.32

TOTAL: 1285.8 100.0 1275.8 100.0 1265.8 100.00

The fat variables are as follows:

Variables:

1. Van den Bergh Food ' s Fluid Flex™ Usage level 7.78%.

2. Calgene's High Stearate Canola Oil. Usage level 7.78%

3. Calgene's Oil with emulsifier (Oil 65 % , EC-25™ 28 % , Dur-Em 114K ™ 7%). Usage level 7.78%.

4. Calgene's Oil with lower emulsifier levels (Oil 73.75%, EC-25™ 21% , Dur-Em 114K™ 5.25%). Usage level 7.78%.

5. Calgene's Oil with lowest emulsifier levels (Oil 82.5 % , EC-25™ 14% , Dur-Em 114K ™3.5%). Usage level 7.78%.

6. Calgene's Oil with emulsifier levels in no. 3 above. Usage level 7.05 % ,

7. Calgene's Oil with emulsifier levels in no. 3 above. Usage level 6.32%.

Emulsifier was placed in oil and heated to 170°F, mixed homogeneous, and cooled to

room temperamre prior to use.

Method

I . Place dry ingredients into Hobart mixing bowl.

2. Add oil (or shortening).

3. Add 60% of water.

4. Mix on speed 1 for 30 seconds.

5. Scrape bowl and paddle.

6. Mix on speed 2 for 4 minutes.

7. Scrape bowl and paddle.

8. Add 20% of water.

9. Mix on speed 1 for 30 seconds.

10. Scrape bowl.

I I . Mix on speed 2 for 2 minutes.

12. Scrape bowl.

13. Add last 20% of water.

14. Mix on speed 1 for 30 seconds.

15. Scrape bowl.

16. Mix on speed 2 for 2 minutes. 17. Pour 425 grams of batter into greased, floured, 4xxxx tinned. 8" steel pans

lined with parchment paper.

18. Tap filled pans on counter ten times to deaerate.

19. Bake cakes until done (internal temperamre over 200 °F). Vulcan convection

oven at 325 °F for 25 minutes, low fan speed, high load setting.

20. Remove from oven.

21. Cool for 30 minutes.

22. Remove cake from pan.

23. Cooled cakes were sealed in plastic bags.

24. Cakes were measured for volume with a layer cake measuring template. A ACC

Method 10-91.

25. Cakes were tested for compression resistance with a TA-XT2 texmre analyzer

on days 1, 3, 5 and 7.

The results are shown in Fig. 3:

RESULTS / DATA

Variable No. Cake Volume Index Bauer Density. ε/100mi

1 74.5 81.52

2 69.5 116.84

3 104.5 53.36

4 103.4 62.96

5 108.0 88.48

6 111.0 56.04

7 107.0 57.36

CΔKF COMPRESSION RESISTANCE (s)

Variable No. Day 1. Day 3. Day 5 Day 7

1 949 1402 1827 1992

2 1376 2279 2850 2946

3 372 541 640 772

4 490 595 730 790

5 541 743 824 919

6 399 579 626 661

7 374 551 651 665

DISCUSSION

1. Softness of cakes made with Fluid Flex was about midway between those made

with unemulsified Calgene stearate oil and those made with emulsified Calgene stearate oils throughout the experiment.

2. Crumb softness of cakes made with the emulsified Calgene stearate oils fell

within a fairly narrow range at the oil and emulsifier levels used in this test.

3. The cake volume index was significantly higher for all the emulsified Calgene

oils.

Results are illustrated in Fig. 3. The cakes made with unemulsified strucmred

stearate oils (Calgene oils) were harder than cakes made with "Fluid Flex. " However,

the emulsified strucmral stearate oils produced softer cakes. Crumb softness of cakes

made with emulsified strucmred stearate oils fall within a fairly narrow range at the oil

and emulsifier levels used. Cake volume index was significantly higher for all the

emulsified strucmred oils.