rexresearch.com
Peggy TOMASULA, et
al.
Casein Food Packaging
Edible, water-resistant, & 500% better
protection vs oxidation
http://www.ars.usda.gov/is/pr/2010/100120.htm
January 20, 2010
Potential of Dairy-Based Package Wraps
Outlined
by Rosalie Marion Bliss
Food-packaging products made from dairy ingredients could provide
a viable alternative to petroleum-based packaging products,
according to a chapter written by Agricultural Research Service
(ARS) scientist Peggy Tomasula for a new book, “Dairy-Derived
Ingredients: Food and Nutraceutical Uses.”
The book was written by an international team of contributors and
published by London-based Woodhead Publishing in October 2009. It
serves as a guide to new developments for the dairy and
nutraceutical industries, as well as researchers in those fields.
Tomasula works at the ARS Eastern Regional Research Center (ERRC)
in Wyndmoor, Pa., where scientists are developing strong,
biodegradable dairy-based films that are better oxygen barriers
than petrochemical-based films. Tomasula leads the center’s Dairy
Processing and Products Research Unit.
Most food packages are made of multilayer films that are thin,
continuous sheets of synthetic polymers. But consumers and food
retailers are concerned about the waste generated during the
manufacture of such packaging. Many, it seems, are interested in
replacing petroleum-based packaging with biobased packaging.
Tomasula’s chapter in the new book is titled “Using Dairy
Ingredients to Produce Edible Films and Biodegradable Packaging
Materials.” The chapter focuses on films made from dairy proteins,
with an emphasis on those based on casein and whey, the major
proteins found in milk. It also covers research efforts to improve
the proteins' mechanical and barrier properties so that these
natural materials eventually could be used in a variety of future
applications.
As a dairy ingredient, casein shows good adhesion to different
substrates. But while casein is an excellent barrier to oxygen,
carbon dioxide, and aromas, it is a weak barrier to moisture.
Because the water-soluble nature of those proteins poses a
challenge, much of the research on edible casein films to date is
directed toward improving their water-vapor-barrier properties.
https://www.sciencedaily.com/releases/2016/08/160821093046.htm
Edible food packaging made from milk
proteins
At the grocery store, most foods -- meats, breads, cheeses, snacks
-- come wrapped in plastic packaging. Not only does this create a
lot of non-recyclable, non-biodegradable waste, but thin plastic
films are not great at preventing spoilage. And some plastics are
suspected of leaching potentially harmful compounds into food. To
address these issues, scientists are now developing a packaging
film made of milk proteins -- and it is even edible.
The researchers are presenting their work today at the 252nd
National Meeting & Exposition of the American Chemical Society
(ACS).
"The protein-based films are powerful oxygen blockers that help
prevent food spoilage. When used in packaging, they could prevent
food waste during distribution along the food chain," says
research leader Peggy Tomasula, D.Sc.
And spoiled food is just one issue. Current food packaging is
mainly petroleum-based, which is not sustainable. It also does not
degrade, creating tons of plastic waste that sits in landfills for
years.
To create an all-around better packaging solution, Tomasula and
colleagues at the U.S. Department of Agriculture are developing an
environmentally friendly film made of the milk protein casein.
These casein-based films are up to 500 times better than plastics
at keeping oxygen away from food and, because they are derived
from milk, are biodegradable, sustainable and edible. Some
commercially available edible packaging varieties are already on
the market, but these are made of starch, which is more porous and
allows oxygen to seep through its microholes. The milk-based
packaging, however, has smaller pores and can thus create a
tighter network that keeps oxygen out.
Although the researchers' first attempt using pure casein resulted
in a strong and effective oxygen blocker, it was relatively hard
to handle and would dissolve in water too quickly. They made some
improvements by incorporating citrus pectin into the blend to make
the packaging even stronger, as well as more resistant to humidity
and high temperatures.
After a few additional improvements, this casein-based packaging
looks similar to store-bought plastic wrap, but it is less
stretchy and is better at blocking oxygen. The material is edible
and made almost entirely of proteins. Nutritious additives such as
vitamins, probiotics and nutraceuticals could be included in the
future. It does not have much taste, the researchers say, but
flavorings could be added.
"The coatings applications for this product are endless," says
Laetitia Bonnaillie, Ph.D., co-leader of the study. "We are
currently testing applications such as single-serve, edible food
wrappers. For instance, individually wrapped cheese sticks use a
large proportion of plastic -- we would like to fix that."
Because single-serve pouches would need to stay sanitary, they
would have to be encased in a larger plastic or cardboard
container for sale on store shelves to prevent them from getting
wet or dirty.
In addition to being used as plastic pouches and wraps, this
casein coating could be sprayed onto food, such as cereal flakes
or bars. Right now, cereals keep their crunch in milk due to a
sugar coating. Instead of all that sugar, manufacturers could
spray on casein-protein coatings to prevent soggy cereal. The
spray also could line pizza or other food boxes to keep the grease
from staining the packaging, or to serve as a lamination step for
paper or cardboard food boxes or plastic pouches. The U.S. Food
& Drug Administration recently banned the perfluorinated
substances that used to coat these containers, so casein coatings
could be a safe, biodegradable alternative.
Bonnaillie says her group is currently creating prototype film
samples for a small company in Texas, and the development has
garnered interest among other companies, too. The group plans to
keep making improvements, and she predicts this casein packaging
will be on store shelves within 3 years.
Peggy M Tomasula
Dairy and Functional Foods
Research Leader
Peggy.Tomasula@ars.usda.gov
Phone: (215) 233-6703
Fax: (215) 233-6795
USDA,REE,ARS,NAA,ERRC
600 E MERMAID LANE
WYNDMOOR, PA, 19038-8598
https://www.ars.usda.gov/research/publications/publications.htm?SEQ_NO_115=327816
Advances in food packaging films from
milk proteins
Technical Abstract: Most commercial petroleum-based food packaging
films are poor oxygen barriers, do not biodegrade, and some are
suspected to even leach compounds into the food product. For
instance, three-perfluorinated coatings were banned from
convenience food packaging earlier this year. These shortcomings
are a problem particularly with high-fat foods, which may solvate
leached compounds faster and tend to oxidize. Packaging films made
from milk proteins are excellent oxygen barriers, up to 500 times
better than LDPE, and completely food-safe. In addition, they are
hydrophilic and repel grease, can be eaten with the food product,
and dissolve easily in hot or cold water. For these reasons,
milk-based films are ideal candidates to coat convenience food
packaging; layer between synthetic films to block oxygen; coat
foods to preserve them and carry additional nutrients; or, form
increasingly-popular single-serve pouches, which can be either
eaten or dissolved, generating zero waste. This presentation
reports ARS' recent advances in strengthening casein-based, edible
packaging films, to physically protect food products, as well as
mediate their hydrophilicity to customize their resistance to
environmental conditions and/or rate of dissolution and enable a
broad range of applications, from cheese-stick wrappers to healthy
cereal glaze. The rheological, mechanical, thermal, structural,
barrier and functional properties of solvent-cast casein-based
films and coatings are characterized using state-of-the art,
environmentally-controlled instrumentation such as DMA-RH,
vapor-sorption analysis (VSA), oxygen permeability analysis, water
vapor transmission, microscopy, and more. Due to the complex,
charged, 3-D structure of protein monomers, casein films are
sensitive to many formulation and processing parameters, including
the caseinate type (calcium or sodium) and concentration,
polysaccharide cross-linkers, alkalinity of the suspension,
film-casting parameters, and the environmental conditions during
drying, storage and testing conditions are critical to the
mechanical properties and shelf-life of these hygroscopic,
versatile edible polymers.
https://www.ars.usda.gov/research/publications/publications.htm?SEQ_NO_115=319019
Electrospinning of caseinates to
create protective fibrous mats
Tomasula, Peggy, Bonnaillie, Laetitia Ana, Sousa, Liu, Linshu
Technical Abstract: JUSTIFICATION Electrospinning is a nonthermal
process that produces fibers with diameters on the micron- or
nano-scales from a polymer solution. If produced by
electrospinning of biopolymer solutions, fibrous mats may be
created for protecting foods, improving food quality and allowing
for the preservation and controlled release of bioactives for
health and wellness. However, little information is available on
electrospinning of food-grade biopolymers. OBJECTIVE To create
fibers for food use from electrospinning aqueous solutions
containing calcium (CaCAS), sodium caseinate (NaCAS), or CAS with
a polysaccharide, such as Pullulan (PUL), and examine the
structure of the fibrous mats created for controlled release of
bioactives. METHODOLOGY An electrospinning unit was used to
generate the fibers at 50 deg C using from 11 to 23 KV and flow
rates from 0.4-3.0 mL/h. The electrospinning apparatus consisted
of a syringe pump leading to a needle; a high voltage source at
the needle with range from 0 to 50 kV; and, a drum cylinder
wrapped in aluminum foil for collection of the fibrous mats. CAS
or CAS:PUL solutions were added to the syringe prior to
experiments. The morphologies of the fibrous mats were determined
using scanning electron microscopy equipped with software to
sample 100 of the constituent fibers to calculate mean diameters.
RESULTS/DISCUSSION Fibers were not produced by electrospinning 5,
10, or 15% (w/w) aqueous solutions of either CAS, possibly because
of little interaction among the CAS, but were produced when the
solutions of either CAS were blended with PUL. PUL forms
entanglements in solution and served as a carrier for CAS.
Electrospinning of neat 15% PUL solutions resulted in fibers with
mean diameters of 190 +/- 50 nm while electrospinning of CaCAS:PUL
solutions resulted in fibers within the range of pure PUL. This
indicated interaction between CaCAS:PUL since larger fibers would
be expected if CaCAS was located on the surface of PUL. CONCLUSION
This is the first example of CAS nano- and micro-fibers prepared
using a polysaccharide carrier, rendering a new dairy product with
potential use in food and packaging applications. Electrospinning
at temperatures ranging from 25 to 50 deg C preserves the
activities of most bioactives embedded in the fibers.
https://www.ars.usda.gov/research/publications/publications.htm?SEQ_NO_115=312048
Biodegradable bioplastics from food
wastes
Authors : Tomasula, Peggy, Liu, Linshu
Technical Abstract: An estimated 1.8 billion tons of waste are
created annually from food processing in the US, including the
peels, pulp, and pomace (PPP) generated from fruits and vegetables
when they are converted into frozen or canned products or pressed
into juice. PPP currently is sold as animal feed at low cost, but
a large portion is discarded at an additional cost. The profitable
use of food processing waste requires a strategy to enhance the
competitiveness of the US food industry and cost effective ways to
protect the environment from contamination. We have developed a
method to make biodegradable bioplastics from food wastes in
combination with other biodegradable materials such as poly
(lactic acid) (PLA), a biodegradable material derived from
fermentation of biomass. The bioplastics may be produced using
existing equipment employed by the plastics industry. The
resulting bioplastics possess mechanical properties that make them
strong enough for use as food containers. They have enhanced water
resistance and are totally biodegradable. Other examples of
byproducts from food processing include the residue from corn
ethanol fermentation, known as distiller’s dried grains with
solubles (DDGS). The production of DDGS has increased from 2.7
million tons in 2000 to 32.5 million tons in 2010. Experts predict
that DDGS will soon outpace their consumption rate as animal feed.
To convert DDGS to bioplastics is an attractive way to overcome
this obstacle and is currently under research in our laboratory.
https://www.youtube.com/watch?v=wt32GgQGTcI&feature=youtu.be
Edible, Biodegradable Food Packaging
Most foods come wrapped in plastic packaging. This type of
packaging creates a lot of waste and aren't that great at
preventing spoilage. Researchers are now developing a
biodegradable film made from milk proteins to hopefully solve
these problems.
US6379726
Edible, water-solubility resistant casein masses
Inventor(s): TOMASULA PEGGY, et al.
An edible, water-resistant composition that can be formed into
shaped articles including film comprises a water-resistant solid
composition of casein. The casein composition may be directly
precipitated from a solution under high pressure treatment with
carbon dioxide. The composition does not have to be crosslinked,
but takes advantage of the natural water-insolubility of the
protein backbone of the casein. The casein composition may be
combined with edible or inert flexibilizers to improve film
properties, and the film may be used to protect food products or
food compositions, yet provide moisture protection. The film of
casein material may exhibit water-insolubility in deionized water
at 20° C. of less than 25% by weight after two hours of immersion
of the film in the deionized water, with or without mild
agitation.
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to the field of casein materials,
casein compositions, and methods of manufacturing casein in an
edible form that is also resistant to dissolving in neutral or
basic aqueous systems.
2. Background of the Art
Casein comprises a group of proteins that forms about 80 percent
of the total proteins in cow's milk. It solidifies when milk is
made slightly acidic and is the chief ingredient in cheese. Casein
is used as a food supplement, an adhesive, and a finishing
material for paper and textiles. It is also used in water paints.
Consumer demands for both higher quality and longer shelf-life
foods have stimulated edible film research. The environmental
movement has promoted increased concern about reducing disposable
packaging amounts and increasing packaging recyclability, farther
contributing to the recent surge in edible coating and film
research. Edible films and coatings are capable of offering
solutions to these concerns by regulating the mass transfer of
water, oxygen, carbon dioxide, lipid, flavor, and aroma movement
in food systems. Edible coatings function by direct adherence to
food products; whereas, edible films act as stand-alone sheets of
material used as wrappings, low moisture baked products, and
intermediate and high moisture foods all exhibit potential for
improvement through the use of edible coatings and films. Dried
foods (e.g., dried vegetables and dried meats) and low moisture
baked products (e.g., crackers, cookies and cereals) are
particularly susceptible to moisture uptake from the atmosphere.
Low moisture baked foods are also susceptible to moisture uptake
from moist fillings and toppings. Such changes can result in loss
of sensory acceptability of the food product, as well as a reduced
shelf-life. Many dried and baked products are also susceptible to
oxidation, lipid migration and volatile flavor loss.
Intermediate moisture foods, such as raisins and dates, often
become unacceptable due to moisture loss over time. Moisture loss
is particularly problematic when the moisture transfers into lower
moisture components of a food system, For example, raisins can
lose moisture to the bran in raisin bran. Nut meats, another
intermediate moisture food, are susceptible to lipid oxidation
resulting in the development of off flavors. High moisture food
components typically lose moisture to lower moisture components.
One classical example of this phenomenon occurs when pizza sauce
moisture migrates into the crust during storage, resulting in a
soggy crust. Oxidation and flavor loss are also problematic to
high moisture food systems. The respiration rates of whole fruits
and vegetables often dictate their shelf lives. Minimally
processed fruits and vegetables are often subject to unacceptable
levels of oxidative browning. Individual food products within the
broad food categories discussed above require different barrier
properties in order to optimize product quality and shelf-life.
Edible films and coatings are capable of solving the barrier
problems of these and a variety of other food systems. See,
Kester, et al., Food Technol. 40:47-59 (1986) and Krochta, in
Advances in Food Engineering, CRC Press, Inc., Boca Raton, Fla.
Singh and Wirakartakusumab (Eds.) p. 517-538 (1992).
Edible films and coatings based on water-soluble proteins are
typically water-soluble themselves and exhibit excellent oxygen,
lipid and flavor barrier properties; however, they are poor
moisture barriers. Additionally, proteins act as a cohesive,
structural matrix in multicomponent systems to provide films and
coatings having good mechanical properties. Lipids, on the other
hand, act as good moisture barriers, but poor gas, lipid, and
flavor barriers. By combining proteins and lipids in emulsion or
bilayer barriers, the advantages of each component can be
exploited to form an improved film system. Plasticizer addition
improves film mechanical properties. It would be desirable to be
able to provide water-insoluble protein films, even if they do not
necessarily provide oxygen barriers.
The harvesting of casein from milk utilizing either acid or an
enzyme precipitation while efficient for recovering the casein
protein from the milk, does not recover any whey protein. After
acid or enzyme precipitation of casein from milk, normally the
whey fraction is discarded. This thus constitutes a waste of some
of the protein content of the milk, even though the utilization of
the whey has improved over the years. It has been suggested as,
for instance, Phillips, et al. U.S. Pat. No. 4,218,490, to harvest
the whey protein content of milk utilizing ion exchange resins.
U.S. Pat. No. 4,545,933 entitled Hydrolyzed Protein Composition
and Process Utilized in Preparation Thereof describes a process
for hydrolyzing casein protein utilizing caustic solutions of
sodium or potassium hydroxide. The hydrolyzed protein produced by
the process of this patent has certain unique properties which are
useful in the preparation of certain processed food products. The
starting materials suggested for the process of U.S. Pat. No.
4,545,933 is acid precipitated casein, that is casein which
contains no whey protein.
The properties of composite bilayer films and coatings have been
studied in the past. Cohesive bilayer films and coatings are often
difficult to form and delamination may occur over time.
Furthermore, bilayer film and coating formation often requires the
use of solvents or high temperatures, making production more
costly and less safe than aqueous emulsion film production.
Protein-lipid emulsion film and coating systems can be formed from
aqueous solutions and applied to foods at room temperature.
Water-insoluble edible films and coatings offer numerous
advantages over water-soluble edible films and coatings for many
food product applications. Increasing levels of covalent
crosslinking in water-insoluble edible films and coatings result
in better barriers to water, although not necessarily barriers to
oxygen, carbon dioxide, lipids, flavors and aromas in food
systems. Film mechanical properties are also improved. Many foods,
such as fruits and vegetables, are exposed to water during
shipping and handling. In these cases, water-insoluble films and
coatings remain intact; whereas, water-soluble films and coatings
dissolve and lose their barrier and mechanical properties. Edible
films in the form of wraps, such as sandwich bags, also require
water-insolubility.
Prior to this invention, water-soluble, protein-based edible films
and coatings have been formed from aqueous solutions of proteins
(Gennadios, et al., in Edible Coatings and Films to Improve Food
Quality, Technomic Publishing Co., Lancaster, Pa., Krochta,
Baldwin and Nisperos-Carriedo (Eds.), Chapter 9, (1994)); however,
a means to produce water-insoluble films and coatings from aqueous
solutions with improved barrier properties had not been
discovered. Carmelization and/or Maillard browning reactions had
been exploited for the formation of improved protein-based oxygen
barrier coatings for fruits and vegetables (Musher, U.S. Pat. No.
2,282,801). Protein thiol-disulfide interchange and free thiol
oxidation reactions had been studied previously (see Donovan, et
al., J. Food Sci. and Technol. 11:87-100 (1987) and Shimada, et
al., J. Agric. Food Chem. 37:161-168 (1989)). However, the use of
these reactions for the formation of new and improved edible
barriers had not been explored. Edible moisture barrier coatings
had been formed out of protein-based aqueous emulsions (see,
Adams, et al., EP 0 465 801 Al and Ukai, et al., U.S. Pat. No.
3,997,674). However, methods for the formation of water-insoluble
protein-based films and coatings had not been discovered.
Others have studied the interactions between proteins and lipids
at interfaces in emulsions and colloidal systems. See, Barford, et
al., in Food Proteins, American Oil Chemists Society, Kinsella and
Soucie, eds., (1989) and Le Meste, et al., in Interactions of Food
Proteins, American Chemical Society, Washington, D.C., Parris and
Barford, eds., (1991). However, regulation of mass transfer as a
function of lipid particle size and distribution in films has not
been explored.
What is needed is a method for preparing water-insoluble
protein-based edible films and coatings from casein that exhibit
improved water-solubility resistance, and barrier and mechanical
properties.
Industrial application of casein, which is the major component of
milk proteins, has been studied in many fields. Although some of
these studies are actually practiced in industries, the amount of
casein used in these applications is limited. The reason is that
casein is difficult to be molded, extruded, or worked, and its
physical properties are limited by its water-solubility. These
physical limitations are in part a result of the fact that this
protein easily forms a stable micelle structure due to its
macromolecular surface activity.
Casein protein is a phosphoprotein possessing a macromolecular
surface activity which enables the protein to form a micelle
structure. The micelle structure renders casein stable in milk.
When separated from milk, casein will form globular micelles if an
alkaline earth metal, such as calcium or magnesium, is present.
Such globular micelles are difficult to disperse in a medium and
difficult to mold.
There have been several proposals for dispersing casein, however,
none brought about good results. For example, Japanese Patent
Laid-open (kokai) No. 138145/1987 discloses a method of dissolving
caseinate in an ethanol aqueous solution and making films by fluid
spreading (which might be a casting method). However, caseinate
becomes too hard when heated in a drying step, preventing it from
being formed into fibers or films. This method therefore has not
been industrially successful. Journal of Japan Agrichemical
Society, 61, 1087-1092 (1987) proposes a method of dissolving or
dispersing casein molecules by breaking down the micelle structure
in a casein solution. The method involves treating the casein
solution with a chelating resin, thereby removing the metals, e.g.
calcium, from the casein solution. The report states that after
the treatment, the casein molecules form sub-micelles, but does
not describe how the sub-micelles can be used.
Casein protein is a water-soluble macromolecular surfactant
consisting of hydrophobic protein and hydrophilic phosphoric acid
groups, the latter groups rendering the protein more soluble in
acid solutions. The phosphoric acid groups are bonded to
counterions, i.e. metals such as calcium and magnesium, which
induce the protein to form globular micelles. This configuration
makes casein molecules difficult to orient in the longitudinal
direction necessary to obtain an acceptable fiber. Obtaining
fibers and other molded articles with acceptable mechanical
properties from casein, therefore, has not been successful.
The properties and structure of the casein molecule include
characterizations as millions of particles/cc with an average path
of 0.37 micrometers between particles. The Molecular Weight varies
from 10 to 3280 million, and the shape of the molecule is likely
spherical. The Composition Size of micelles depends on initial
s:k-casein (sigma/kappa) ratio, absolute protein concentration and
Ca++ concentration. If micelles are removed by
ultracentrifugation, the sediment is a clear gel. When dispersed
in water, get an opaque colloidal suspension. It is highly
hydrated, with about 2.5 grams water/gm. protein. All casein
subunits are accessible to high molecular weight reagents and the
association of subunits is through noncovalent bonds.
The principal solid constituent of milk is casein, a protein. When
milk is allowed to stand in a warm place, it sours, and the casein
is precipitated by the action of lactic acid bacteria. The thick
precipitate, or curd, is separated from the thin, watery residue
known as whey. Today curd is usually prepared with rennet, which
acts to speed the separation process. The next steps in the making
of cheese are salting (for flavor and eventually to aid in curing)
and pressing (to shape the cheese and eliminate more whey). The
curd is then ready for curing and is stored under temperature- and
humidity-controlled conditions for varying lengths of time. In
general, the longer the curing or aging process, the more
pronounced the flavor of the finished product. During curing,
gases are formed within the cheese, and in some types they are
unable to escape; this produces the holes characteristic of some
cheeses. To aid the curing process, harmless blue-mold spores are
introduced into the blue-veined cheeses (Roquefort or blue
cheese), and white-mold spores are sprayed on the surface of such
cheeses as Brie and Camembert. This produces a rind, which may be
eaten. Cheese casein is not a preferred source of casein in the
practice of the present invention.
Casein may be derived from any original source and may be prepared
by conventional methods can be used as the raw material in the
present invention without any specific limitations. Casein is the
principal protein in milk (whether, fresh cow milk, fresh goat
milk or non-fat dried milk, ultrafiltered milk, other available
milk forms and sources, and exists in milk as a colloidal
aggregate of protein together with phosphorus and calcium. Other
metal ions, such as magnesium, also may be present. Casein most
useful in the present invention may be precipitated from milk by
the addition of CO2 to cause precipitation.
In an optional initial series of steps in the present method, the
casein may be treated to remove some of the calcium and any other
forms of metals which may be present, although some calcium must
remain to provide some structure to the film. This may be
accomplished by any method effective for selectively removing
metal ions, such as, for example, ion-exchange or chelation. A
preferred method of removing the metals comprises contacting an
aqueous solution or dispersion of casein with a chelating agent.
Any chelating agent may be used in the method, however resins
having fixed chelating functional groups are preferred. Chelating
resins having iminodiacetic acid groups as functional groups are
most preferably used as the chelating resin. A method for removing
metal ions from casein using a chelate-functional resin which is
described in said report of Journal of Japan Agrichemical Society,
supra. Preferably, the carboxyl terminals of the iminodiacetic
acid groups in the resin comprise hydrogen ions (H<+>).
H-type resins are preferred because in order to ensure complete
removal of metal ions from the casein, therefore the absence of
alkali metals such as sodium at the functional group terminal is
imperative. An iminodiacetic acid functional resin which is useful
in the present invention, for example, is Uniselex.(TM). UR30
(trademark, manufactured by Unitica Co., Ltd.). This step is
carried out by contacting an aqueous casein solution with the
chelating resin under conditions appropriate to remove some all of
the metal ions from the casein solution, thereby forming
sub-micellular casein. Sub-micellular casein thus obtained may be
dried by a conventional method, if desired.
According to the present invention casein, which has been
heretofore difficult to form into molded articles such as fibers,
films, or the like, can be easily molded and manufactured into
regeneration films, and it is readily predictable that fibers and
other articles made of natural casein proteins may be made from
the materials of the present invention. Casein fibers can be woven
into cloth or sheets which are useful for various applications. In
addition, because the raw material (casein) is naturally found in
milk, articles made according to the invention that may be used as
food materials, such as edible fibers. Casein is a naturally
occurring material which is biodegradable, therefore, articles
made from casein fibers or films contribute to global environment
conservation. Casein films, non-woven fabrics of casein fibers or
woven fibers and yarns, for example, could be used to make
biodegradable packaging materials.
In an attempt to improve the structural stability of articles made
from starch-based compositions, other ingredients have been
included in the formulations. For example, compositions have been
developed that include starch in combination with a
water-insoluble synthetic polymers. Unmodified starches have also
been combined with protein to provide moldable, biodegradable
thermoplastic compositions. For example, Nakatsuka et al. (U.S.
Pat. No. 4,076,846; issued Feb. 28, 1978) discloses an edible
binary protein-starch molding composition containing a salt of a
natural protein (i.e., casein), an unmodified, high amylose starch
material, an edible plasticizer (i.e., sorbitol), and a lubricant
(i.e., a fatty acid polyol ester), and having a final water
content of about 10-40%. The composition is molded, for example,
by extrusion through a die, into an article having a water content
of about 5-30 wt-%. A disadvantage of these starch-based plastics
is that the molded articles made from such compositions have a
high tendency to absorb water, which causes the articles to lose
mechanical strength and to disintegrate quickly.
U.S. Pat. No. 5,543,164 describes a method of forming an edible,
protein-based water-insoluble film by treating a solution of the
protein to effect disulfide formation and a denatured protein
solution, then forming the denatured protein solution into a film.
The denaturing is effected by causing a thiol-disulfide exchange
by heat treatment and/or chemical reaction, e.g., heating between
70 and 95 degrees Celsius for up to three hours to initiate
disulfide crosslinking reactions. These relatively high
temperatures are essential for enabling the crosslinking to occur.
The casein referred to here is probably calcium or sodium
caseinate, usually manufactured by adding calcium or sodium
hydroxide to acid casein (e.g., manufactured by the HCl process)
and heated to at least about 75 degrees C. This type of calcium
caseinate will have approximately the same molar proportions of
casein with natural calcium therein, but the calcium in the
calcium caseinate does not hold the micelles together as occurs
with the natural calcium. The use of the vacuum is to assist in
the removal of air bubbles that tend to get trapped in film after
mixing, pouring or other mechanical procedures.
Conventional concentrating processes depend upon direct chemical
treatment of the source vegetable matter to concentrate the
protein. For example, raw soy products such as soy meal, soy
flakes, and soy flour are treated with acid (e.g., hydrochloric
acid) to precipitate protein and separate the protein from whey,
sugars, oils and proteins which will not precipitate. Some of the
acid remains in the protein precipitate and must be removed by
additional processing either specific or generic to removal of the
acid residue. As the acid is undesirable from many standpoints of
flavor, aesthetics and health, it is desirable that in at least
some uses that substantially all of the acid (reduced to an acid
level of less than 0.5% by weight) is removed. The processing
necessary to do this may be sufficiently harsh as to reduce the
value and content of the soy protein concentrate or soy protein
isolate produced by the acid treatment process.
There are also many physical processes for producing protein-rich
products from grains. U.S. Pat. No. 5,135,765, for example,
describes a process for producing a protein-rich product from
brewer's spent grain containing germ, husks and a proteinaceous
material. The process requires the use of high water content spent
grain (e.g., at least about 65% water by weight), passing the wet
spent grain through a mill to press and grind the solids, and then
sieving the spent grain in water to produce an at least 50% by
weight protein product. After formation of the first concentrate,
the coarse fraction may be extracted with alkaline aqueous
solution at elevated temperature to form an extract, and the
extract is acidified to form a further concentrated protein rich
precipitate.
It is well known that soy bean products may have undesirable taste
components. These components are known to be reduced or removed by
selection of unique varieties of soy beans for the original
source, heating an intermediate soy bean product to reduce
lipoxygenase, extraction with an aqueous solution, extraction with
an alkali solution, extraction with a reducing agent (e.g., see
U.S. Pat. No. 5,023,104), extraction with organic solvents (e.g.,
removal of chlorohydrins from hydrolyzed protein compositions in
U.S. Statutory Registration No. H989) and extraction with high
pressure or supercritical carbon dioxide (e.g., "Preparation and
Evaluation of Supercritical Carbon Dioxide Defatted Soybean
Flakes" A. C. Eldridge, et al., Journal of Food Science, Vol. 51,
No. 3, 1986, pp. 584-587; "Off-Flavor Removal from Soy-Protein
Isolate by Using Liquid and Supercritical Carbon Dioxide" JAOCS,
Vol. 72, no. 10, 1995, pp. 1107-1115; "Emulsifying Properties of
Low-fat, Low-cholesterol Egg Yolk Prepared by Supercritical CO2
Extraction" Journal of Food Science, Vol. 61, No. 1, 1996, pp.
19-23 and 43; and U.S. Pat. No. 4,493,854 shows extraction of oil
from soy (e.g., flakes) by CO2 extraction, leaving extracted meal
as a by-product. The purpose of the process is to improve the
flavor of the soy products by removal of undesirable flavor
materials in the soy product. The process in U.S. Pat. No.
4,493,854 produces an enhanced flavor oil by tempering the initial
soy material with moisture and extracting oil from the tempered
soy product, but the by-product of protein and other solids is not
a significantly concentrated product and is not an isolate, as it
would still contain the whey, sugars and other materials not
extracted by the CO2.
Bovine milk contains about 3 to 4% protein. The casein component
of the bovine milk protein constitutes about 80% of the total
protein. The remaining protein is divided among certain whey
proteins with the principal one being .beta.-lactoglobulin.
It was recognized in antiquity that the casein protein of bovine
milk could be separated from the "whey" fractions by in situ
acidification of milk utilizing enzyme extracts or by the direct
addition of acid to the milk. For the preparation of casein from
milk, after skimming the cream off the top the milk is acidified
either by the addition of acid or by an enzyme. Below about pH 4.7
the casein precipitates as "curd" leaving a clear liquid, the
"whey".
In order to improve the heat sealability of edible films and
thereby overcome the above-described disadvantages, a number of
methods have been proposed. They include the method of forming a
film from an intimate blend of amylose, an alkali metal salt of
casein, and a low-molecular-weight plasticizer (Japanese Patent
Laid-Open No. 112533/'76); the method of dipping a collagen film
in, or coating it with, a mixture of gelatin or glue and a
plasticizer (Japanese Patent Laid-Open No. 11280/'77); the method
of forming a film by laminating a polysaccharide with gum arabic,
pullulan, starch or gelatin (Japanese Patent Laid-Open No.
76336/'85); and the method of incorporating a solid fat in an
edible film (Japanese Patent Laid-Open No. 59855/'88).
However, the films formed from an intimate blend of amylose, an
alkali metal salt of casein, and a low-molecular-weight
plasticizer, the films formed by laminating a polysaccharide with
gum arabic, pullulan or starch, and the edible films having a
solid fat incorporated therein still fail to exhibit adequate heat
sealability. The films formed by laminating a collagen film or a
polysaccharide with gelatin show a marked improvement in heat-seal
strength, but have the disadvantage that the presence of gelatin
in the surface layer causes severe blocking of films and this
makes it difficult to handle the films.
SUMMARY OF THE INVENTION
An edible, water-resistant composition that can be formed into
shaped articles including film comprises a water-resistant solid
composition of casein. The casein composition may be directly
precipitated from a solution under high pressure treatment with
carbon dioxide. The composition does not have to be crosslinked,
but takes advantage of the natural water-insolubility of the
protein backbone of the casein. The casein composition may be
combined with edible or inert flexibilizers to improve film
properties, and the film may be used to protect food products or
food compositions, yet provide moisture protection.
DETAILED DESCRIPTION OF THE INVENTION
The terms water-insoluble, and substantially water-insoluble have
specific meanings within the practice of the present invention.
The term "water-insoluble" means that less than 2% of total weight
of casein-based material is dissolved from a mass after immersion
in deionized water at 20 degrees Celsius for two hours. The term
"substantially water-insoluble" means that less than 10% of total
weight of casein-based material is dissolved from a mass after
immersion in deionized water at 20 degrees Celsius for two hours.
The term "marginally water-insoluble" means that less than 25% of
total weight of casein-based material is dissolved from a mass
after immersion in deionized water at 20 degrees Celsius for two
hours. "Casein-based" refers to materials within the film or
composition that are casein or chemically derived from casein.
Some materials of the invention may display substantial water
insolubility of less than 15% of total weight of casein-based
material being soluble or dissolved from a mass after immersion in
deionized water at 20 degrees Celsius for two hours. Some
materials of the invention may display water insolubility of less
than 10% or less than 5% of total weight of casein-based material
being soluble or dissolved from a mass after immersion in
deionized water at 20 degrees Celsius for two hours. A material is
casein-based if at least forty percent or at least fifty percent
of its mass is casein. Usually the mass of the material is from
about 45-90% casein (or casein-based material), more usually from
about 50 to 80% by weight casein (or casein-based material),
usually still more than 80%, or more than 85%, 90% or 95% casein
(or casein-based material).
The process of the invention may be practiced to produce the novel
casein materials (e.g., as a film) by using an initial pressure
above the surface of the solution that is usually provided as a
pressure of from about 400 to 1800 pounds per square inch (psi) to
the solution/dispersion. If the pressure is increased to above
2000 psi for an extended period of time, the resulting
casein-based product tends to be water-soluble. This is theorized
to be a result of removal of Ca, Mg, PO4, or citrate groups that
normally bind the micelle together. The film may comprise a film
of micelles in the integral structure of a film. The initial
pressure in the vessel will usually be lower and the pressure may
be increased at a desired rate. There is likely to be at least
some CO2 present in the gas over the surface of the solution in
the vessel, but normal atmospheric CO2 content would not be
sufficient to effect the process of the present invention. The
initial solution may also be pretreated to advance the process.
For example, prior to the application of pressure in the vessel or
even before introduction of the solution into the vessel, the
solutions may be pretreated by heating the solution (e.g., from
about 30 to less than 65 degrees Centigrade). The resulting
supernatants would then be chilled before the CO2 treatment. These
are examples of advantageous but not essential types of
pretreatment steps in the practice of the present invention.
Sufficient CO2 should be introduced into the system to lower the
pH below 7, preferably below about 6 and more preferably below
about 5.5. Heating is performed either before, during or after the
addition of the CO2 into the system, or at a combination of these
times. The heating is generally effected to provide a solution
temperature of between 30 and less than 65[deg.] C. (e.g., 30 to
60[deg.] C.) for casein protein and may be varied as desired or
applicable on an individual basis for the particular protein
source selected. This heating also increases the pressure within
the reaction vessel or system and assists in interactions between
compounds.
Additional CO2 may be added, reducing the pH of the solution
further, often by at least 0.5 pH units. The CO2 may be added to
effect a supercritical state over the solution to assure the
effectiveness and concentration levels of the carbonic acid in the
solution. The final pH is generally below 5.5, more often above
5.0, as for example between 4.5 and 5.5 or between 5.0 and 5.3
(e.g., 5.1 or 5.2). The solution in the vessel or the solution
within a continuous apparatus system is then held at these
conditions for a time sufficient to assist in the solubilization
of the protein.
The pressurized solution is then depressurized, removing the whey.
The casein is removed from the reactor. Casein is then dried. The
solution, when coated out and dried, forms a substantially
water-insoluble film of casein. The coating out of the solution
may be by casting, extruding, molding or other forming processes.
It is important to note that the present invention uses the
carbonic acid in the solution to precipitate the protein in the
solution during the pressurizing and heating. This is
substantively different than the use of supercritical CO2 to
remove trace flavor materials as practiced in mere extraction
processes. Extraction removes either desirable materials from a
mass (so that the desirable materials, such as oils, may be
collected) or removes minor amounts of undesirable materials from
a mass (such as the removal of objectionable flavors from soy, as
described above). Extraction processes, in fact, are usually
performed on concentrates and isolates and reference is seldom if
ever made to any further concentration of the solids, even though
some minor increase of the percentage of protein in the solid
product is likely to occur. Additionally, these extraction
processes often act to remove materials which are soluble in the
CO2 rather than act to precipitate materials by a process where
after adding the flakes, meal or flour to water there is a
dissolution of the globulins and albumins into the water of the
solution or dispersion.
When CO2 is added to the milk, there is a drop in pH due to the
formation of carbonic acid from the CO2. The drop in pH causes a
change in the solubility of the casein, precipitating from the
solution, while whey proteins remain in the solution/dispersion.
However, upon release of pressure, the pH returns almost to the
original value of the solution/dispersion before introduction of
the CO2, and indication that most of the CO2 has evolved.
Therefore there are no contaminating salts in the product.
With regard to copending, commonly assigned application processes
such as the whey process described in U.S. patent application Ser.
No. 08/996,136, whey proteins are comprised of alpha-lactalbumin
(alpha-La, about 30%) and beta-lactalbumin (about 50%), the rest
being immunoglobulins (Igs), Bovine serum albumin (BSA), and
proteose-peptones. An enriched fraction of alpha-La containing the
alpha-La, Igs, BSA and proteose-peptones was isolated. The
mechanism appears to be a combination of pH, heat, and possibly
salt formation. The pH is initially lowered with CO2 and probably
causes a release of calcium from the alpha-La and changes the
conformation of the protein. The calcium probably exists in
solution as a bicarbonate. Addition of heat above 50[deg.] C.,
along with the depressed pH causes the alpha-La to form
aggregates. The alpha-La most likely entraps the Igs, BSA and
maybe the proteose-peptones. The aggregates may not necessarily
get big enough to drop out of the whey solution/dispersion as a
precipitate, so centrifugation or filtration (e.g.,
microfiltration) may be needed to remove them in this or a later
step. In this case the heat does seem to foster aggregation, and
may change the mechanical strength of the protein.
In U.S. Pat. No. 5,432,265, the casein precipitation process was
used with CO2 to demonstrate the fact that the process can operate
under high pressure continuously. The present invention
establishes that the apparatus described therein can be used as
part of the present process. In the process of U.S. Pat. No.
5,432,265, the component being removed is casein, a protein
product comprising proteins linked by calcium phosphate bonds. One
of the first steps in the process of U.S. Pat. No. 5,432,265 is to
break these bonds so that individual proteins are held in
solution/dispersion. That process then adjusts the temperature of
the solution/dispersion, causing the proteins to agglomerate,
which may entrap some small amount of other solids and dissolved
materials within the network of agglomerated proteins. This
process is specifically temperature dependent and the proteins
precipitate as agglomerated materials. In the present invention,
there is little or no dissolving of calcium phosphate bonds to
free proteins, there is little or no agglomeration of proteins,
proteins precipitate by more traditional physical phenomena where
the change in pH of the solution/dispersion causes decreased
solubility of selected proteins, and those specific proteins
(which fortuitously happen to be the desirable proteins)
precipitate from the solution/dispersion, leaving other dissolved
and carried materials within the solution/dispersion. The CO2
controls the Ca-phosphate bonds rather than completely eliminating
them.
With regard to the general extraction process patents described
above, these mechanisms rely upon the differences in density
between the oils and CO2. At supercritical pressures of around
10,000 psi, CO2 has a density and other properties that mimic
those of a liquid solvent. At supercritical pressures of around
10,000 psi, CO2 has a density that mimics a liquid solvent. The
supercritical CO2 also exhibits transport properties, such as
viscosity and diffusivity, that mimic a gas. In operation, the
practitioners typically pack a very small column with soy flakes
(or other material), pressurize with CO2, circulate the CO2
through the column for a couple of hours to dissolve oil and
establish equilibrium, and then crack open a valve to a flask. The
rapid decrease in pressure causes the CO2 to gasify and the oil
previously carried by the CO2 to precipitate into the flask. The
flakes don't move continuously through the process, whereas in the
present invention, where a continuous process would be performed,
all solids and liquids would move. More importantly, in the
extraction process, only the oil and essentially hydrocarbon
soluble materials are absorbed into the supercritical gas stream,
but there is no precipitation of protein from a
solution/dispersion.
As used herein, unless otherwise noted, the wt-% of the components
of the composition are based on the total dry weight of the
composition.
Other Additives
Plasticizers. Preferably, the composition before molding
may include about 9-20% water to provide a plasticizing effect to
facilitate processing, preferably about 9-11 wt-% water for
compression molding, or up to about 20 wt-% water for extrusion or
injection molding. According to the invention, the molded article
will contain about 4-5 wt-% water. In addition to water, a minor
but effective amount of a compatible plasticizer may also be
included in the composition to facilitate processing and increase
flexibility of the molded article. It has been found that
inclusion of a plasticizer such as glycerol, tends to increase
flexibility, but decrease the tensile strength and increase the
water absorption of the molded article. Therefore, it is preferred
that, where desired, the composition include a minor amount of
plasticizer of about 0.1-40 wt-%, preferably about 5-35 wt-%,
based on the total solids weight of the composition. When using
glycerol as the plasticizer, for example, the preferred range is
between 20 and 30% by weight of plasticizer. For extrusion and
injection molding, the composition may include up to about 45 wt-%
plasticizer.
Plasticizers that may be used according to the invention, alone or
in combination, include low molecular weight hydrophilic organic
compounds such as di- or polyhydric alcohols and derivatives
thereof, as for example, glycerol, glycerol monoacetate, diacetate
or triacetate, polyglycerol, glycerol monostearate, sorbitol,
sorbitan, mannitol, maltitol, ethylene glycol, diethyl glycol,
propylene glycol, polyvinyl alcohol, and the like; sodium
cellulose glycolate, cellulose methyl ether, and the like;
triethyl citrate, and the like; and polyalkylene oxides such as
polyethylene glycols, polypropylene glycols, polyethylene
propylene glycols, polyethylene glycol fatty acid esters, and the
like. Preferred plasticizers according to the invention are
glycerol, glycerol monoacetate, glycerol monostearate, and
polyglycerol. But plasticizers are preferred that are edible
themselves, such as glycerol, mannitol, sorbitol, maltitol, gum
arabic, and the like.
Lubricants. The composition may further contain a minor but
effective amount of a lubricating agent to provide a lubricating
effect, for example, by aiding in the release of the molded
article from surfaces, to facilitate extrusion, and the like.
Water-insoluble lubricants may also increase the water-resistance
of the products. Examples of suitable lubricants that may be used
in the compositions, either alone or in combination with another
lubricant, include mono- and diglycerides, and fatty acids,
preferably saturated fatty acids; phospholipids such as lecithin;
phosphoric acid-derivatives of the esters of polyhydroxy
compounds; vegetable oil, preferably hydrogenated forms; animal
lipids, preferably hydrogenated forms to prevent thermal
oxidation; and petroleum silicone and mineral oils. The amount of
lubricant contained in the composition is preferably about 5 wt %
or less, 2 wt-% or less, and more preferably about 0.1-1 wt-%,
based on the total solids weight of the composition.
Extenders. Extenders, for example, water soluble
polysaccharides such as methylcellulose, hydroxymethylcellulose,
microcrystalline cellulose and cellulose fiber, and synthetic
polymers such as poly(acrylic acids), poly(methacrylic acids),
poly(vinyl acetates), poly(vinyl alcohol), and poly(vinyl acetate
phthalate), may also be included in the composition. Preferably,
an extender is included in the composition in an amount of about
<50 wt-%, more preferably about 3-20 wt-%, based on the total
solids weight of the composition.
Preservatives. A compatible antimicrobial agent such as a
fungicide or bactericide may also be included in the composition
in an amount effective to prevent growth of fungi, bacteria and
the like, in or on the compositions or an article formed from the
compositions. The antimicrobial agent should not induce
undesirable interactions or chemical reactions between the
components of the composition.
Antioxidants. The compositions may include a compatible
antioxidant to retard oxidation and darkening of color of the
composition during processing, such as by extrusion or molding at
elevated temperatures. Suitable antioxidants include, for example,
butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT),
Irganox 1010, propyl gallate (PG), .alpha.-tocopherol (Vitamin E),
and ascorbic acid preferably in the form of ascorbyl palmitate,
and the like. The composition may include about 0.001-1% of an
antioxidizing agent, preferably about 0.01-1%, preferably about
0.1-0.5%.
Colorants. The compositions may further include a coloring
agent. Coloring agents, suitable for use in the present
compositions include, for example, azo dyes such as Bismarck Brown
2R and Direct Green B; natural coloring agents such as
chlorophyll, xanthophyll, carotene, and indigo; and metallic
oxides such as iron or titanium oxides. The coloring agent may be
included in the composition at a concentration of about 0.001 to
10 wt-%, preferably about 0.5 to 3 wt-%, based on the total solids
weight of the composition.
Edible Compositions. The present casein-based compositions
may be comprised entirely of ingredients that may be consumed at
nontoxic levels by a human or other mammal. In that case, the
article formed from the composition would be biodegradable as well
as edible by a mammal. An edible composition according to the
present invention, would comprise, for example, the casein protein
combined with a compatible and edible solvent such as an aqueous
alcohol or mildly alkaline aqueous solution (pH 8), and optional
additives including, for example, a plasticizing agent such as
glycerol, a lubricating agent such as lecithin and mono-or
di-glycerides, an extender such as microcrystalline cellulose or
cellulose fiber, an antioxidant such as ascorbic acid, and/or an
antimicrobial agent such as methylparaben. An effective amount of
an edible flavoring agent such as cocoa, vanillin, fruit extracts
such as strawberry and banana, and the like, may also be included
to enhance the taste of an edible composition. The composition may
also be nutritionally reinforced, as for example by the inclusion
of vitamins or minerals. The composition may also be ground and/or
pelletized and used as animal feed.
Any food component may be coated, wrapped, packaged, sealed,
skinned, cased or the like in the composition of the present
invention with moisture resistance provided. The food component
may comprise meat(s), vegetable(s), fruit(s),(supplement(s),
fiber(s), vitamin(s) or mixtures thereof. The food component may
be cooked, partially cooked, or raw. Ground meats comprising at
least one of beef, lamb, turkey, chicken, pork and the like (with
flavoring or dilutants added) are particularly suitable for use
with the present invention.
Cucumber, carrot, spinach, cabbage, Japanese konjak and the like
can be singly or taken together as the raw material of the
bound-formed food of the present invention. Vegetables and fruits
are, however, used very often together with proteinous raw
materials, which results in their value added.
Furthermore, any solid foods such as bean, biscuit, cracker,
caramel, chocolate, cake, rice snack, potato chip, cookie, pie,
candy and the like can be raw food material for the production of
bound-formed foods of the present invention.
The bound-formed foods produced from these raw materials include
not only bound products of chicken, animal meat, fish meat and the
like, which can be made into steak, and ham, sausage, hamburg
steak, meat balls, Japanese kamaboko, Japanese chikuwa, Japanese
hampen and the like, but also other bound-formed foods which are
not known in the prior art, such as a product having a novel
structure.
Materials and Methods
Materials. Carbon dioxide (CO2)-precipitated casein was prepared
as described previously (Tomasula et al., U.S. Pat. No. 5,432,265,
issued 1995 and J. Dairy Sci., Vol. 78, pp. 506-514, 1995) by
injecting CO2 into milk at 5520 kPa and 38[deg.] C. in a batch
reactor. The reactor contents were held for 5 minutes. After
precipitation, the casein was washed with distilled water to
remove whey proteins, lactose, and minerals. The casein was then
freeze-dried. Alanate 310 calcium caseinate (New Zealand Milk
Products, Inc., Santa Rosa, Calif.) was used to make films for
comparative purposes. Proximate analysis of the caseins was
determined in our laboratory according to methods described
previously (Tomasula et al., U.S. Pat. No. 5,432,265 et al.,
1995). Glycerol (GLY), used as a plasticizer, was purchased from
Aldrich Chemical Co. (Milwaukee, Wis.).
Film-Making Procedure. Aqueous solutions of 2, 4, 6, and 8% (w/w)
CO2-casein and Alanate 310 calcium caseinate were prepared. Twenty
milliliters of each was then pipetted into 100 mm wide*15 mm high
polystyrene Petri dishes (Fisher Scientific Co., Pittsburgh, Pa.)
to cast films of the pure caseins.
Aqueous solutions with total GLY concentration and either Alanate
310 or CO2-casein of 6% (w/w) were then prepared so that the
resulting films contained either 20, 30, 40 or 50% (w/w) GLY. The
solutions were stirred vigorously using a hand-held stirrer for 2
minutes. A light vacuum was applied to each solution to remove
bubbles. Five films were cast from each solution. The films were
allowed to dry overnight at ≈23[deg.] C. and 50% relative humidity
(RH) and then were stored in a desiccator at ≈50% RH and 23[deg.]
C. Storing the films at 50% RH prevents the films from shrinking,
warping, or developing cracks and permits easy removal from the
plates. RH was maintained in the desiccator using a saturated
NaHSO4 solution.
Thicker films were prepared from the same solutions containing
either casein and 30% (w/w) GLY by pipetting ≈28 mL of solution
into a Petri dish and following the film-making procedure
described above.
Film Thickness. A model 3 micrometer (B. C. Ames Co., Waltham,
Mass.) was used to measure film thickness. Reported values of film
thickness are the mean of 10 measurements selected randomly over
the face of the film. The precision of the thickness measurements
was ±5%.
Water Vapor Permeability (WVP) Measurements. The apparatus and
method used to measure WVP have already been described (Parris et
al., J. Agric. Food Chem., Vol. 38(3), pp. 824-829, 1990). The
method is based on ASTM E96-80 (ASTM, 1980) as modified by McHugh
et al. (J. Food Sci., 58:899-903, 1993). Four replicates each of
CO2-casein or Alanate 310 films containing 6% (w/w) total solids
and 30% (w/w) GLY were tested. Air velocity was maintained at 150
m/min across the films. Temperature was controlled at 30±2[deg.]
C. WVP for all films was determined with the shiny side down
facing the vapor source.
Tensile Property Measurements. An Instron model 1122 tensile
tester equipped with a 2000 g load cell was used to measure
tensile strength (TS), elongation to break (ETB), and initial
modulus (IM). Five replicates were run for each film composition
using 5 mm wide specimens. A gauge length of 25 mm and an
extension rate of 5 mm/min were used. Samples were stored at 50%
RH for at least 24 h before testing. Standard deviation was
calculated using version 6.0 of the Instron software. Scatter
plots of the data were prepared using SigmaPlot 4.0 for Windows,
Chicago, Ill.
Solubility Measurements. The procedure used to determine the
solubility of the films in water is similar to that described in
Gontard et al. (J. Food Sci., 57, pp. 190-199, 1992). Water
solubility was determined for CO2-casein and Alanate 310 films
containing 6% (w/w) total solids and either 0 or 30% GLY. A 4 cm
diameter disk was cut from each of the films, weighed, and then
immersed in water at room temperature for 24 h with stirring. The
nondissolved film was then dried at 100[deg.] C. for 24 h and
weighed. The percentage solubility was defined as the mass of
casein in the film that dissolved divided by the initial mass of
casein in the film. The experiments were performed in triplicate.
Scanning Electron Microscopy. Strips of dry films were immersed in
1% glutaraldehyde-0.1 M imidazole-HCl solution at pH 6.8 for 48 h
at room temperature. After washing in imidazole buffer for 1 h,
these strips were immersed in 2% OSO4-0.1 M imidazole solution for
2 h, washed in distilled water, dehydrated in a graded series of
ethanol solutions, and embedded in an epoxy resin mixture. Thin
sections were cut with diamond knives, stained with solutions of
2% uranyl acetate and lead citrate, and examined in a model CM12
scanning transmission electron microscope (Philips Electronics,
Mahway, N.J.) operated in the bright field mode at an instrumental
magnification of 22000*.
Statistical Analyses. Microsoft Excel 97 SR-1 (Microsoft Corp.,
Redmond, Wash.) was used for all statistical analyses. The data
were analyzed with ANOVA, and means were compared using the F
test. Differences were considered to be significant at P<0.05.
Qualitative Film Properties. CO2-casein and calcium caseinate
films prepared from the protein solutions without added GLY were
brittle. CO2-casein films prepared from 2% (w/w) solutions were
brittle and difficult to peel from the Petri dishes. Calcium
caseinate films prepared from either the 2 or 4% (w/w) solutions
were difficult to remove from the dishes. To facilitate comparison
between the properties of the two films, the films were prepared
from solutions containing 6% (w/w) total solids. Films prepared
from 8% (w/w) solutions were qualitatively comparable to films
prepared from the 6% (w/w) casein solutions. Only the calcium
caseinate films were tested for tensile properties; they performed
similarly to the films cast from 6% (w/w) solutions. These films
were not subjected to water vapor barrier property testing or
solubility testing. We limited our study to films prepared from
the 6% (w/w) solutions because films prepared from solutions
containing the least amount of protein are most desirable in
commercial applications to keep costs low.
Films prepared from CO2-casein were slightly milky in appearance,
but transparent. The milky appearance may be due to the presence
of intact casein micelles. Some of the CO2-casein films dried to
almost a matte surface. The calcium caseinate films were
transparent and had smoother surfaces. Added plasticizer did not
affect the appearance of the films. The CO2-casein films appeared
to have more "depressions", which were almost pore-like, compared
to the caseinate films. The surface depressions in both films may
be a result of localized phase separations during drying of the
film. Observation by SEM photographs indicates that the casein
micelles in the calcium caseinate films (A) are large and randomly
distributed throughout the film.
TABLE 1
Proximate Analysis of CO2-Casein and Commercial
Calcium Caseinate (Reported on Moisture-Free Basis)
ash protein fat
lactose
casein (%) (%) (%) (%) Ca (%)
P (%) Na (%)
CO2-casein 3.89 94.1 1.52 0.5
1.6 0.5 0.2
calcium 4.44 92.3 0.5 2.7 1.6
0.3 0.8
calcium 4.3 94.5 1.1 0.1 1.3
0.8 0.2
<a>Alanate 310, New Zealand Milk Products, Inc. (Santa Rosa,
CA).
<b>Analysis performed in our laboratory.
<c>Analysis supplied by the manufacturer.
Casein micelles in the CO2-casein films (B) are much smaller and
located in a more ordered arrangement. The smaller micelles in the
CO2-casein can be attributed to the higher precipitation pH, which
was sufficient to disrupt only some of the larger micelles.
Results of the proximate analyses of the CO2-casein and the
calcium caseinate used in this study are shown in Table 1.
Analytical results obtained in our laboratory for the two caseins
showed equivalent amounts of calcium. Calcium caseinate contains
≈60% as much phosphorus as does CO2-casein.
Tensile Properties. Tensile Properties for blends of CO2-casein
and calcium caseinate films with GLY (average film thickness=0.15
mm) are plotted in FIG. 3. Films containing 0 and 10% GLY were too
brittle for testing. Tensile strength (TS), elongation to break
(ETB), and initial modulus (IM) were determined. ETB is a measure
of the flexibility of the film, and IM is a measure of the
stiffness of the film.
For both films, TS decreased with increasing GLY content. At 20%
(w/w) GLY content, TS was >30% greater for the CO2-casein
films, but the difference in TS decreased with increasing GLY
content. There was no significant difference between the values of
ETB for the CO2-casein films and calcium caseinate films over the
entire range of GLY content. The values of ETB dropped with GLY
content >40%. IM for the CO2-casein films is greater than IM
for the calcium caseinate films over the entire range of added
GLY.
TS results for both films are in general agreement with the values
listed in Chen (J. Dairy Sci., 78, pp. 2563-2583, 1995) for
caseinate films containing GLY. TS values for other protein film
types are also of similar magnitude (Gnanasambandam et al., J.
Food Sci., 62, pp. 395-398, 1997; Ghorpade et al., supra, 1995).
TABLE 2
Variation of Tensile Properties with Film Thickness for CO2-Casein
and Calcium Caseinate Films Containing 30% (w/w) Glycerol
film thickness TS* ETB* IM*
film type (mm) (MPa) (%) (MPa)
CO2-casein 0.11 1.2<a> 50.2<a>
9.6<a>
0.15 3.0<b> 74.2<a> 40.9<b>
calcium caseinate 0.11 1.6<ac>
66.6<a> 10.8<a>
0.15 1.9<c> 76.0<a> 8.9<a>
*Within each category, means with no superscript in common
are significantly different (P < 0.05). TS = Tensile Strength.
ETB = Elongation To Break. IM = Initial Modulus.
TABLE 3
WVP Values of CO2-Casein and Calcium Caseinate Films
Containing 30% (w/w) Glycerol
average film WVP*
film type thickness (mm) RH (%) swelling
(g.mm/kPa.h.m<2>)
CO2-casein 0.112 85.8 No
2.22<a>/(1.90)
0.163 87.7 No 2.58<b>/(2.22)
0.184 87.9 No 3.21<c>/(2.80)
0.277 89.7 No 3.80<d>/(3.41)
Ca caseinate 0.171 86.5 Yes 3.18<c>
0.222 85.5 Yes 4.45<e>
*Values in parentheses for the CO2-casein films were
calculated without the water vapor permeability (WVP) correction
factor of McHugh et al. (1993). Within each category, means with
no superscript in common are significantly different (P <
0.05).
The differences in the tensile properties of the two films,
especially at lower GLY content, may be related to the manner in
which calcium and phosphorus are bound to the caseins. CO2-casein
is precipitated at pH 5.4 (Tomasula et al., U.S. Pat. No.
5,432,265 et al., 1995) instead of the isoelectric pH 4.6 used to
isolate acid casein. The higher precipitation pH is associated
with higher calcium content. Because aggregates are formed at this
pH, it is assumed that some of the micellar calcium phosphate,
which maintains the casein micelle structure, is dissolved in the
whey. In acid casein manufacture, most of the micellar calcium
phosphate dissolves. Commercial calcium caseinate is made by
dissolving acid casein in water followed by the addition of
calcium hydroxide to replace calcium. The casein coagulate is
broken down upon addition of calcium hydroxide, weakening
hydrophobic protein interactions. CO2-casein film may be stronger
because more of the micellar calcium and phosphate linkages are
intact. It was concluded that the functional properties of
CO2-casein differ from those of calcium caseinate most likely
because of the manner in which calcium and phosphorus are
associated with the caseins (Strange et al., J. Dairy Sci., 81,
pp. 1517-1524, 1998).
The flexibility of the films is not significantly different over
the entire range of GLY content. ETB for the CO2-casein film
declined with GLY content >30% and for the calcium caseinate
film declined with GLY content >40%. GLY reduces intermolecular
forces in films by inserting itself between the protein chains.
There may be an electrostatic attraction between calcium and the
hydroxyl groups of GLY. GLY may also establish hydrogen bonding
with amino acid residues of casein.
Tensile properties of polymeric films are not affected by film
thickness. In our study, small but significant differences in TS
and IM were noted with increasing film thickness, as shown in
Table 2. For the CO2-casein films, TS increased slightly with
increasing film thickness. ETB for both film types did not vary
with the change in film thickness. IM for the CO2-casein film
increased with film thickness but did not vary significantly for
the calcium caseinate film. Differences are most likely an
artifact of making and drying the films in Petri dishes and
possibly would not be observed if the film were made in a
different manner. All films had the same surface area exposed to
50% RH, but the thicker films took longer to dry, as expected. As
the films were drying, it was observed that film formed and
adhered to the sides of the Petri dishes while the rest of the
film was reduced in height by evaporation. The height of the film
was approximately evenly reduced from its center to its perimeter.
The films shrank away from the sides of the dish when dry, with
some puckering observed at the edges of the films. This drying
pattern may have affected the distributions of protein, glycerol,
and water molecules, leading to the small differences in measured
tensile properties.
The tensile properties of the casein films may be considered
moderate (10-100 MPa) at the lower end of added GLY, in comparison
to low-density polyethylene films (Krochta and De Mulder-Johnston,
Food Techno., 51, pp. 61-74, 1997). ETB is close to that observed
in oriented polypropylene films with ETB of 60%.
Water Vaper Permeability (WVP). WVP was determined for CO2-casein
and calcium caseinate films containing 30% (w/w) GLY. The values
are reported in Table 3 for various film thicknesses. The WVP
correction factor (McHugh et al., 1993) was used to correct for
the effect of the water vapor partial pressure gradient in the
stagnant air layer of the test cup. WVP for the CO2-casein films
was also calculated using ASTM Method E96 with the assumption of
100% RH (values shown in parentheses). WVP values for the
CO2-casein films were less than those for the calcium caseinate
films at a particular film thickness. As shown in Table 3, the
increased WVP for calcium caseinate films is accompanied by a
smaller RH inside the test cup. The decreased RH is due to
absorption of water by the protein, resulting in swelling of the
film. CO2-casein films did not swell-an indication of greater
resistance to moisture mass transfer. Differences in the WVP
properties may be attributed to the more rigid structure of the
CO2-casein films.
Both films show an increase in WVP with increasing thickness that
is indicative of hydrophilic films (McHugh et al., supra, 1993;
Ghorpade et al., Trans, ASAE, 38, pp. 1805-08, 1995). However, the
effects are not as pronounced as they are in McHugh et al. (Supra,
1993) because the films in this study are thicker and the
resulting RH covers a narrow range. WVP values are more likely a
result of structural differences. WVP is not a function of film
thickness for hydrophobic films.
WVP values reported in this study are greater than those reported
by Avena-Bustillos and Krochta (J. Food Sci., 58, pp. 904-907,
1993) for calcium caseinate films with approximately half the
thickness.
Water Solubility. Water solubility was determined for various
casein films, with and without 30% added GLY plasticizer. Results
are reported in Table 4. Calcium caseinate films were easily
dispersed in water. Upon dispersal in water, the CO2-casein-based
films showed no loss of integrity but changed from a transparent
film to white and developed some tackiness. The whitening of the
films is because of the clustering of the casein proteins due to
the repulsion of water molecules. When the films were vigorously
stirred, they broke up but did not dissolve further. The films did
not whiten during the WVP studies, though.
TABLE 4
Water Solubility of CO2-Casein Films and Comparison to Other
Protein Films
protein film water solubility (%) reference
CO2-casein 7.1 this study
CO2-casein-30% GLY 16.8 this study
calcium caseinate 90.0 this study
calcium caseinate-30% GLY 100 this study
soy film 37.7 Ghorpade et al. (1995)
rice bran films Gnanasambandam, et. al.
(1997)
pH 9 11
pH 3 9
The increased solubility for the films containing GLY appears to
be due to the presence of the plasticizer, because both films have
about the same protein content. The presence of the plasticizer,
in the range of 0-30% GLY, does not appear to significantly
increase the solubility of the protein as seen by Stuchell and
Krochta (1994) for edible soy films or by Mahmoud and Savello (J.
Dairy Sci., 76, pp. 29-35, 1993) for whey films.
The results are compared to the solubilities of other protein film
types reported in the literature in Table 4. The water solubility
of the CO2-casein film is comparable to that for rice bran films
with GLY added as plasticizer (Gnanasambandam et al., 1997).
Casein produced by sparging (scattering the bubbles during
introduction) CO2 into milk forms strong films that are highly
hydrophobic. The properties are most likely a result of
precipitation at a higher pH, which leaves some of the micellar
calcium phosphate structure intact, and the higher precipitation
temperature, which may increase protein-protein interactions as
well. CO2 films with added GLY plasticizer are stronger and
stiffer than similar calcium caseinate/GLY films but have lower
WVP and water solubility. The hydrophobic nature of these films
may recommend them for uses that require better strength and
moisture resistance.