Note: Descriptions are shown in the official language in which they were submitted.
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A PROTEIN RICH FOOD INGREDIENT FROM BIOMASS AND
METHODS OF PRODUCTION
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation-in-part of application serial no.
14/809,051, filed
July 24, 2015, and claims the benefit of U.S. provisional application no.
62/029,324, filed
July 25, 2014, each of which are hereby incorporated by reference in their
entireties,
including all Tables, Figures, and Claims.
Background
[0002] Proteins are essential nutritional components and protein rich
material is often
added to various types of food products in order to increase the nutritional
content. Current
sources of protein material include various plant, grain, and animal sources,
but their
availability is often subject to wide seasonal fluctuations, the weather, crop
failures, and other
unpredictable factors therefore limiting their commercial use by food
manufacturers. Grain
based solutions for protein production also consume a large amount of
productive land and
water resources that might otherwise be better utilized. These sources are
also limited in their
ability to supply sustainable supplies of proteins in the quantities
necessary. Additional and
more reliable sources of proteins are needed to supply both a growing humanity
and as feed
for domestic animals.
[0003] Algal and microbial sources of proteins or other nutritional
materials have great
potential and would be highly desirable as they can reduce seasonal
fluctuations and
nevertheless provide a consistent, economic, and sustainable source of
nutritional materials to
food providers. Proteins and other nutritional materials produced by these
sources could be
used to supplement cereals, snack bars, and a wide variety of other food
products.
Furthermore, if organisms dependent on photosynthesis for energy (e.g., algae)
could be
made to produce useable proteins, it would have a highly favorable effect on
the energy
equation in food production.
[0004] However, algal and microbial sources of proteins often suffer from
significant
disadvantages in that they contain substances that are severely displeasing in
terms of their
organoleptic taste and smell properties. It would be highly advantageous to be
able to harvest
proteins from algal and/or microbial organisms that do not have the
displeasing organoleptic
properties. Such proteins would be very useful as foods, food ingredients, and
nutritional
supplements.
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Summary of the Invention
[0005] The present invention provides methods for producing a proteinaceous
food or
food ingredient from a sustainable, economic, and stable source such as
microbial biomass.
The methods generally involve the pasteurization of the biomass followed by a
acid wash
step where the pH of the biomass is lowered to a pH of less than 4.5 and held
at such pH for
at least 10 minutes. Additional steps can also be utilized such as a
delipidation steps and/or a
mechanical homogenization step. The methods produce a protein composition that
has
pleasing organoleptic properties. Also provided are methods of making a food
product or
food ingredient, and method of improving the organoleptic properties of a
proteinaceous
composition. Also provided is a proteinaceous food or food ingredient having
acceptable
organoleptic taste and smell properties.
[0006] In a first aspect the invention provides methods of producing a
protein composition
from cellular biomass. The methods involve performing a pasteurization step on
cellular
biomass to produce a pasteurized biomass, subsequent to step a), exposing the
pasteurized
biomass to acidic conditions by adjusting the pH of the biomass to a depressed
pH of 4.5 or
less, or less than 4.5 and holding the pH of the biomass at said depressed pH
for at least 10
minutes to convert the proto-protein into the protein composition. The acid
conditions can be
a depressed pH of less than 4.0 and the pH of the microbial biomass is held at
said depressed
pH for at least 20 minutes. In one embodiment the pH of the biomass is
adjusted to about 3.5
and the pH is held for about 30 minutes. In another embodiment after adjusting
the pH to the
depressed pH of less than 4.0 the pH is adjusted to a raised pH of greater
than 4Ø In yet
another embodiment after adjusting the pH to the depressed pH of less than 4.0
the pH is
adjusted to a raised pH of greater than 4Ø The biomass can be exposed to the
acidic
conditions by contacting the biomass with an inorganic acid. In various
embodiments the
inorganic acid can be sulfuric acid or hydrochloric acid.
[0007] In some embodiments the methods further involve performing a
delipidation step
on the cellular biomass. The methods can improve the organoleptic properties
of a protein
composition in the cellular biomass. The delipidation step can be performed
after the
pasteurization step and before the biomass is exposed to the acidic
conditions, and the
biomass is delipidated by subjecting it to mechanical homogenization while in
contact with a
solvent. The solvent can be, but is not limited to, any one of ethyl alcohol,
isopropyl alcohol,
and a mixture of hexane and acetone. The pasteurization step can involve
exposing the
biomass to a temperature of at least 55 C for at least 20 minutes. The
cellular biomass can
be algal biomass.
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[0008] In another aspect the invention provides methods of making a food
product. The
methods involve combining the protein material produced by a method of the
invention with
a food or food ingredient to make said food product. The food or food
ingredient can be
made according to any of the methods described herein. The food product can
be, but is not
limited to, a breakfast cereal, a snack bar, a soup or stew, a nutrition bar,
a binder for bulk
artificial meats, an artificial cheese. The food product can also be animal
feed.
[0009] In various embodiments less than 25% of the protein molecules have a
molecular
weight of below 15 kD. The method can also decrease the ratio of arginine,
glutamic acid, or
hydroxyproline comprised in the protein material relative to the ratio in the
delipidated
biomass.
[0010] In some embodiments the methods can also involve a step of
centrifugation and the
production of a centrifugation pellet and supernatant, wherein the ratio of
arginine in the
pellet/supernatant is less than 1.0, or wherein the ratio of glutamic acid in
the
pellet/supernatant is less than 1Ø
[0011] In another aspect the invention provides a food ingredient. The food
ingredient can
be a protein material derived from cellular biomass according to a method of
the invention,
and the protein material can have at least 65% protein content (w/w); less
than 6% lipid
content (w/w); and less than 8% ash content. The lipids can be fatty acids,
which can be
polyunsaturated fatty acids. In one embodiment the cellular biomass is algal
biomass and the
algal protein composition contains at least 75% protein w/w and less than 5%
lipid content
w/w. The food ingredient can be produced in the form of a powder.
[0012] In another aspect the invention provides methods of producing a
protein material.
The methods involve exposing a delipidated biomass that contains a proto-
protein to acidic
conditions by adjusting the pH of the biomass to a depressed pH of less than
4.5 and holding
the pH of the biomass at said depressed pH for at least 10 minutes to convert
the proto-
protein into the protein material. In one embodiment the pH of the biomass can
be adjusted
to a depressed pH of less than 4.0 and the pH of the biomass is held at said
depressed pH for
about 30 minutes, but in other embodiments the pH of the biomass is adjusted
to about 3.5
and the pH is held for about 30 minutes. In one embodiment after adjusting the
pH to the
depressed pH of less than 4.0 the pH is adjusted to a raised pH of greater
than 4.0, but in
another embodiment after adjusting the pH to the depressed pH of less than 4.0
the pH is
adjusted to a raised pH of about 4.5.
[0013] In some embodiments less than 25% of the proto-protein molecules
have a
molecular weight of below 15,000 daltons. The methods of the invention can
also decreases
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the ratio of arginine, glutamic acid (or glutamic acid and glutamine), or
hydroxyproline
comprised in the protein material relative to the ratio in the delipidated
biomass. The
methods can also involve a step of centrifugation and the production of a
centrifugation pellet
and supernatant, which can be done after the exposure to acidic conditions,
and wherein the
ratio of arginine in the pellet/supernatant is less than 1.0 and/or wherein
ratio of glutamic acid
in the pellet/supernatant is less than 1Ø
[0014] In another aspect the invention provides methods of improving the
hedonic
properties of a protein containing composition by subjecting the protein
containing
composition to a method of the present invention.
Description of the Figures
[0015] FIG. 1 is a flow chart showing steps than can be used in various
embodiments of
the methods of the invention. Not all steps need be included in every
embodiment of the
methods. The steps can be performed in the order shown in Figure 1, or in a
different order.
[0016] FIG. 2 is a bar graph illustrating the percentages of the named
amino acids
contained in a dried protein concentrate product from methods including an
acid wash step
versus methods not including an acid wash step.
[0017] FIG. 3 is a bar graph illustrating the removal of lipidic material
at steps of a
process of the invention.
Detailed Description of the Invention
[0018] The invention provides a stable and sustainable source of a
proteinaceous food or
food ingredient. The source of the food or food ingredient can be biomass
produced by
microbial biomass. Non-limiting examples of microbes that can be used to
product the
biomass include phototrophic and/or heterotrophic algae, kelp, and seaweed.
The organisms
can be either single cellular or multi-cellular organisms. These exemplary
sources have great
potential as a stable and sustainable source of proteinaceous food or food
ingredients. The
invention therefore discloses protein materials useful as food, food
ingredients, or food
supplements and which have high nutritional value and acceptable or pleasing
organoleptic
taste and smell properties. Also disclosed are methods of manufacturing the
food ingredients
and methods of manufacturing food products containing a food ingredient of the
invention.
[0019] The invention provides a proteinaceous material that is useful as a
food or food
ingredient. A "proteinaceous" material can have a protein content of at least
50% or at least
60% or at least 65% or at least 68% or at least 70% or at least 72% or at
least 75% or at least
78% or at least 80% or at least 85% or at least 90%, or from 50% to 70%, or
from 65% to
75%, or from 70% to 80%, or from 70% to 85%, or from 70% to 90%, or from 75%
to 90%,
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or from 80% to 100%, or from 90% to 100%, all w/w. In various embodiments the
food or
food ingredient contains all amino acids essential for humans and/or domestic
animals and/or
pets or any mammal. In some examples the animals can be cattle, swine, horses,
turkeys,
chickens, fish, or dogs and cats.
[0020] The proteinaceous food or food ingredient can have varied lipid
content such as,
for example, about 5% lipid or about 6% lipid or about 7% lipid, or about 8%
lipid or less
than 8% or less than 7% or less than 6% or less than 5% lipid or less than 4%
lipid or less
than 3% lipid or less than 2% lipid or less than 1% lipid or less than 0.75%
lipid or less than
0.60% lipid or less than 0.5% lipid or from about 1% to about 5% lipid or from
2% to about
4% lipid. In different embodiments non-protein nitrogen content can be less
than 12% or less
than 10% or less than 8% or less than 7% or less than 6% or less than 5% or
less than 4% or
less than 3% or less than 2% or less than 1% or from about 1% to about 7% or
from 2% to
about 6% in the proteinaceous food or food ingredient. In a particular
embodiment the food
or food ingredient contains at least 80% protein w/w and less than 5% lipid
w/w. The lipid
content of the proteinaceous food or food ingredient can be manipulated as
explained herein
depending on the source of the protein material and the uses of the protein
material to be
produced, as well as by varying the steps in its production. The lipid content
in the food or
food ingredient can be provided, either partially or completely, by
polyunsaturated fatty
acids. The polyunsaturated fatty acids can be any one or more of gamma-
linolenic acid,
alpha-linolenic acid, linoleic acid, stearidonic acid, eicosapentaenoic acid,
docosahexaenoic
acid (DHA), and arachiconic acid, in any combinations. In any of the
compositions the ash
content can be less than 10% or less than 9% or less than 8% w/w.
[0021] The protein material of the invention can be utilized in a wide
variety of foods. It
can be used either as a supplement or a food substitute. As examples, the
protein material
can be utilized or incorporated within cereals (e.g. breakfast cereals
containing mostly grain
content), snack bars (a bar-shaped snack containing mostly proteins and
carbohydrates),
nutritional or energy bars (a bar-shaped food intended to supply nutrients
and/or boost
physical energy, typically containing a combination of fats, carbohydrates,
proteins, vitamins,
and minerals), canned or dried soups or stews (soup: meat or vegetables or a
combination
thereof, often cooked in water; stew: similar to soup but with less water and
cooked at lower
temperature than soup), as a binder for bulk and/or artificial meats
(artificial meats are
protein rich foods, usually based on soy or plant proteins, but having no real
meat of animal
origin in them, but they have characteristics associated with meat of animal
origin), cheese
substitutes, vegetable "burgers", animal or pet feed (e.g. in animal or
livestock feed for
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consumption by domestic animals and/or pets - these feeds can be mostly grain
products),
and much more. It can also be a nutritional supplement such as a protein or
vegetable protein
powder. The protein material can also be converted into a food ingredient,
e.g., a protein rich
powder useful as a substitute for grain-based flour. The protein materials are
useful as food
ingredients or as foods for both human and animal consumers. In addition to
providing an
advantageous source of protein the proteinaceous material of the invention can
also provide
other nutrients, such as lipids (e.g., omega-3 and/or omega-6 fatty acids),
fiber, a variety of
micronutrients, B vitamins, iron, and other minerals being only some examples.
[0022] The algal or microbial organisms that are useful in producing the
biomass from
which the protein material of the invention is obtained can be varied and can
be any algae,
plant, moss, or microbe that produces a desired protein-containing product. In
some
embodiments the organisms can be algae (including those classified as
"chytrids"),
microalgae, Cyanobacteria, kelp, or seaweed. The organisms can be either
naturally
occurring or can be engineered to increase protein content or to have some
other desirable
characteristic. In particular embodiments microbial or algal sources are
utilized. In different
embodiments algae and/or cyanobacteria, kelp, and seaweed of many genera and
species can
be used, with only some examples being those of the genera Arthrospira,
Spirulina,
Coelastrum (e.g., proboscideum), macro algae such as those of the genus
Palmaria (e.g.,
palmata) (also called Dulse), Porphyra (Sleabhac), Phaeophyceae, Rhodophyceae,
Chlorophyceae, Cyanobacteria, Bacillariophyta, and Dinophyceae. The algae can
be
microalgae (phytoplankton, microphytes, planktonic algae) or macroalga.
Examples of
microalgae useful in the invention include, but are not limited to,
Achnanthes, Amphiprora,
Amphora, Ankistrodesmus, Asteromonas, Boekelovia, Bolidomonas, Borodinella,
Botrydium,
Botryococcus, Bracteococcus, Chaetoceros, Carter/a, Chlamydomonas,
Chlorococcum,
Chlorogonium, Chlorella, Chroomonas, Chrysosphaera, Cricosphaera,
Crypthecodinium,
Cryptomonas, Cyclotella, Dunaliella, Elhpsoidon, Emil/an/a, Eremosphaera,
Ernodesmius,
Euglena, Eustigmatos, France/a, Fragilaria, Fragilariopsis, Gloeothamnion,
Haematococcus
(e.g., pluvial/s), Halocafeteria, Hantzschia, Heterosigma, Hymenomonas,
Isochrysis,
Lepocinclis, Micractinium, Monodus, Monoraphidium, Nannochloris,
Nannochloropsis,
Navicula, Neochloris, Nephrochloris, Nephroselmis, Nitzschia, Ochromonas,
Oedogonium,
Oocystis, Ostreococcus, Parachlorella, Parietochloris, Pascheria, Pavlova,
Pelagomonas,
Phceodactylum, Phagus, Picochlorum, Platymonas, Pleurochrysis, Pleurococcus,
Porphyridium, Prototheca, Pseudochlorella, Pseudoneochloris,
Pseudostaurastrum,
Pyramimonas, Pyrobotrys, Scenedesmus (e.g., obliquus), Schizochlamydella,
Skeletonema,
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Spyrogyra, Stichococcus, Tetrachlorella, Tetraselmis, Thalassiosira,
Tribonema, Vaucheria,
Viridiella, Vischeria, and Vo/vox.
[0023] The
cells or organisms of the invention can be any microorganism of the class
Labyrinthulomycetes. While the classification of the Thraustochytrids and
Labyrinthulids
has evolved over the years, for the purposes of the present application,
"labyrinthulomycetes"
is a comprehensive term that includes microorganisms of the orders
Thraustochytrid and
Labyrinthulid, and includes (without limitation) the genera Althornia,
Aplanochytrium,
Aurantiochytrium, Botryochytrium, Corallochytrium, Diplophryids, Diplophrys,
Elina,
Japonochytrium, Labyrinthula, Labryinthuloides, Oblongochytrium, Pyrrhosorus,
Schizochytrium, Thraustochytrium, and Ulkenia. In some examples the
microorganism is
from a genus including, but not limited to, Thraustochytrium,
Labyrinthuloides,
Japonochytrium, and Schizochytrium.
Alternatively, a host labyrinthulomycetes
microorganism can be from a genus including, but not limited to
Aurantiochytrium,
Oblongichytrium, and Ulkenia. Examples of suitable microbial species within
the genera
include, but are not limited to: any Schizochytrium species, including
Schizochytrium
aggregatum, Schizochytrium limacinum, Schizochytrium minutum; any
Thraustochytrium
species (including former Ulkenia species such as U visurgensis, U amoeboida,
sarkariana, U profunda, U radiata, U minuta and Ulkenia sp. BP-5601), and
including
Thraustochytrium striatum, Thraustochytrium aureum, Thraustochytrium roseum;
and any
Japonochytrium species. Strains of Thraustochytriales particularly suitable
for the presently
disclosed invention include, but are not limited to: Schizochytrium sp. (S31)
(ATCC 20888);
Schizochytrium sp. (S8) (ATCC 20889); Schizochytrium sp. (LC-RM) (ATCC 18915);
Schizochytrium sp. (5R21); Schizochytrium aggregatum (ATCC 28209);
Schizochytrium
limacinum (IFO 32693); Thraustochytrium sp. 23B ATCC 20891; Thraustochytrium
striatum
ATCC 24473; Thraustochytrium aureum ATCC 34304); Thraustochytrium roseum(ATCC
28210; and Japonochytrium sp. Ll ATCC 28207. For the purposes of this
invention all of the
aforementioned organisms, including the chytrids, are considered "algae" and
produce "algal
biomass" when fermented or cultured. But any cells or organisms that produce a
microbial
biomass that includes a desired protein can be utilized in the invention. In
some
embodiments the biomass is derived from organisms that produce a protein
composition that
is organoleptically undesirable or unacceptable, which can be to the extent
that a
proteinaceous material derived from the organism is organoleptically
unacceptable as a food
or food ingredient.
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[0024] In still further embodiments the microbial organism can be
oleaginous yeast
including, but not limited to, Candida, Cryptococcus, Lipomyces, Mortierella,
Rhodosporidium, Rhodotortula, Trichosporon, or Yarrowia. But many other types
of algae,
cyanobacteria, kelp, seaweed, or yeast can also be utilized to produce a
protein rich biomass.
These are not the only sources of biomass since biomass from any source can be
used that
contains desired proteinaceous material of significant nutritional value.
Biomass
[0025] Biomass is that biological material derived from (or having as its
source) living or
recently living organisms. Algal biomass is derived from algae, and microbial
biomass is
derived from microorganisms (e.g. bacteria, unicellular yeast, multicellular
fungi, or
protozoa). The term "cellular biomass" indicates algal and/or microbial
biomass. Biomass
utilized in the present invention can be derived from algae, microbes, or any
organism or
class of organisms, including those described herein. Microbial or algal
biomass can be
harvested from natural waters or cultivated. When cultivated, this can be done
in open ponds
or in a photobioreactor or fermentation vessels of any appropriate size. The
microbes or
algae can be either phototrophic or heterotrophic. In some embodiments only
light and
carbon dioxide are provided but nutrients can be included in any culture
medium, for
example nitrogen, phosphorus, potassium, and other nutrients. In other
embodiments sugars
(e.g., dextrose), salts (e.g., Na2SO4, CaC12, (NH4)2SO4), and other nutrients
(e.g., trace
metals) are included in the culture medium depending on the specific needs of
the culture.
[0026] When sufficient biomass has been generated the biomass can be harvested
from
fermentation or cultivation. The harvest can be taken or made into the form of
a broth,
suspension, or slurry. The biomass can generally be easily reduced by
centrifugation to a raw
biomass of convenient volume. Fermentation broth is also easily removed from
the cells by
centrifugation. One or more optional steps of washing the pellet can also be
performed.
Organoleptic Properties
[0027] Organoleptic properties refers to those properties of a food or food
ingredient
relating to the sense of taste and/or smell, particularly with reference to
the taste and/or smell
property being pleasing or unpleasant to a human or animal consumer. Methods
of
evaluating and quantifying the organoleptic taste and/or smell properties of
foods are known
by those of ordinary skill in the art. This evaluation enables one to place a
particular food or
food ingredient, on an organoleptic scale indicating a more or less desirable
taste and/or smell
property relative to another food or food ingredient.
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[0028] Generally these methods involve the use of a panel of several
persons, for example
an evaluation panel of 3 or 4 or 5 or 6 or 7 or 8 or 9 or more than 9 persons.
As further
examples panels can also include 11 or 15 or 19 persons. The panel is
generally presented
with several samples to be evaluated (e.g., 3 or 4 or 5 or 6 or 7 or 8 or more
than 8 samples)
in a "blind" study where the panel members do not know the identity of each
sample. The
samples can be proteinaceous material derived from cellular biomass. The panel
then rates
the samples according to a provided scale, which can have 3 or 4 or 5 or 6 or
more than 6
categories describing the taste and/or smell properties of each sample. The
findings of panel
members (e.g. a majority) can then be utilized to determine whether a food
sample has more
or less desirable organoleptic properties relative to other food samples
provided. The
categories can be correlated to more or less desirable organoleptic properties
and can be
comprised on an organoleptic scale. A sample scoring in one category is
considered to have
more or less desirable organoleptic properties than a sample scoring in
another category. In
some embodiments the biomass or proteinaceous material in the biomass has
unacceptable or
undesirable organoleptic properties, but the organoleptic properties of the
proteinacous
material can be improved by applying the methods described herein. The
proteinaceous
component can include the protein portion and any lipidic or other component
that is
covalently or otherwise closely associated with the protein component as
described herein.
[0029] In some studies a "standard" food or proteinaceous material can be
included to
represent an acceptable organoleptic profile ¨ i.e. taste and smell
properties. Those samples
rating equivalent to or higher than the standard are organoleptically
acceptable or desirable
while those rating lower are unacceptable or undesirable. In various
embodiments the
standard can be soy or whey or pea protein. The organoleptic properties of a
proteinaceous
material derived from biomass can be improved by applying the methods
described herein.
[0030] One example of such a method of evaluating such properties of food
is the 9 point
hedonic scale, which is also known as the "degree of liking" scale. (Peryam
and Girardot,
N.F., Food Engineering, 24, 58-61, 194 (1952); Jones et al. Food Research, 20,
512-520
(1955)). This method evaluates preferences based on a continuum and
categorizations are
made based on likes and dislikes of participating subjects. The 9 point method
is known to
persons of skill in the art, and has been widely used and shown to be useful
in the evaluation
of food products. One can therefore evaluate whether certain foods have more
desirable or
less desirable taste and/or smell properties. Both taste and smell properties
can be evaluated
according to the hedonic scale. In one embodiment the protein food or food
ingredient
produced by the methods of the present invention scores higher on the 9 point
hedonic scale
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versus protein products from the same source that has not been subjected to
one or more steps
of the invention. Other methods of evaluating organoleptic taste and/or smell
properties can
also be utilized.
[0031] The specific criteria utilized by an evaluation panel can vary but
in one
embodiment the criteria include whether the organoleptic properties of a
sample are generally
pleasing or displeasing. Thus, in one embodiment a sample can be rated as
having generally
pleasing organoleptic properties at least equivalent to a standard. Other
common criteria that
can be evaluated include, but are not limited to, whether the sample has a
smell or taste that is
briny (having a salty or salt water character), fishy (having a character
related to fish),
ammonia-like (having a character related to or resembling ammonia). Any one or
more of
these properties can be evaluated. These can be subjective determinations but
people are
familiar with these sensations and, when provided to a panel of persons to
evaluate,
meaningful conclusions are generated. Other criteria that can be used are the
general
organoleptic taste and smell properties of the sample indicated by whether the
sample has
more pleasing, less pleasing, or is about the same as a standard sample
provided.
[0032] Certain chemicals that cause undesirable organoleptic properties are
removed by
the methods described herein. These chemicals can be one or more of a number
of
malodorous and/or foul tasting compounds, which in some cases are volatile
compounds.
Without wanting to be bound by any particular theory examples of compounds
believed to
contribute to undesirable organoleptic properties include lipidic compounds,
including
saturated or unsaturated or polyunsaturated fatty acids (e.g., DHA) and their
breakdown
products, lysophospholipids, aldehydes, and other breakdown products. These
fatty acids or
their breakdown products can also become oxidized (perhaps during isolation
and/or
purification of a proteinaceous material) and such compounds give unpleasant
organoleptic
properties to a food or food ingredient.
[0033] In some embodiments the compounds that confer undesirable organoleptic
properties are lipidic material, which can be covalently bound to desired
proteins or otherwise
closely associated with the protein content of the material. Lipidic compounds
can also be
non-covalently bound but nevertheless closely associated with the protein in
such a way that
they cannot be purified way from the protein by conventional purification
methods. The
chemicals can also be saturated or unsaturated fatty acid moieties. The fatty
acid (or fatty
acid moieties) can comprise but are not limited to gamma-linolenic acid, alpha-
linolenic acid,
linoleic acid, stearidonic acid, eicosapentaenoic acid, docosahexaenoic acid
(DHA), and
arachiconic acid, any w-3 or w-6 fatty acid, a breakdown product of any of
them, or any of
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the aforementioned in an oxidized form. The methods of the invention can
reduce the
amount of one or more of these compounds in the protein material by at least
20% or at least
30% or at least 40% or at least 50% or at least 70% or at least 80% or at
least 90% versus the
amount in protein material from the biomass that has not been subjected to a
method of the
invention. Malodorous and/or foul tasting compounds (organoleptically
unacceptable
compounds) can also include oxidized lipids (e.g., oxidized unsaturated fatty
acids or
oxidized omega-3 fatty acids, for example any of those described above) as
well as proteins
that can confer the malodorous and/or foul tasting properties. Malodorous
and/or foul tasting
compounds can also comprise lipidic material covalently bound to or otherwise
closely
associated with proteins in the proteinaceous material. Chemicals causing
undesirable
organoleptic properties can also be enzymatic or chemical breakdown products
of lipid
molecules, for example any of the lipid molecules described herein.
Methods
[0034] The methods of the invention can comprise any one or more, or all of
the following
steps. The methods can comprise a step of fermentation of cellular biomass,
such as an algae
or micro-algae or microbe; one or more steps of pasteurization; one or more
steps of lysing
and/or homogenization of the cellular biomass, which can be done by any
suitable method
(e.g., mechanical homogenization), and can be done in any of the solvents
listed herein; one
or more steps of delipidation of the cellular biomass, which can be performed
in any suitable
solvent as described herein and can be done simultaneously with or during the
homogenization step; one or more steps of acid washing; one or more steps of
solvent
washing or solvent extraction, using any suitable solvent. Examples of
suitable solvents are
described herein. Solvent washing or solvent extraction can also remove lipid
molecules
(delipidation); additional steps can comprise drying of the cellular biomass;
optionally
passing of the biomass through a particle size classifier; and retrieval of
proteinaceous
material. The methods can involve performing the steps in any order, and one
or more of the
steps can be eliminated. One or more of the steps can be repeated to optimize
the yield or
quality of protein material from the biomass such as, for example, repetition
of one or more
delipidation step.
[0035] The selected biomass can be fermented in a fermentation broth and
conditions
desirable for the type of biomass selected. After fermentation one or more
steps of washing
the pellet can be performed. A step of mechanical homogenization can also be
performed.
This can be done, for example, by bead milling or ball milling, but other
forms of mechanical
homogenization can also be used. Some examples of mechanical homogenization
include,
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but are not limited to, grinding, shearing (e.g., in a blender), use of a
rotor-stator, a Dounce
homogenizer, use of a French press, vortexer bead beating, or even shock
methods such as
sonication. More than one method can be used to homogenize the biomass.
[0036] Pasteurization is a process that destroys microorganisms through the
application of
heat. It is used in a wide variety of food preparation processes.
Pasteurization can involve
heating the biomass mixture to a particular temperature and holding it at the
temperature for a
minimum period of time. The pasteurization step can be accomplished by raising
the
temperature of the biomass to at least 50 C or at least 55 C or about 60 C
or at least 60 C
or at least 65 C or about 65 C or at least 70 C or about 70 C, or from 50
¨ 70 C, or from
55 ¨ 65 C. The mixture can be held at the temperature for at least 10 minutes
or at least 15
minutes or at least 20 minutes or at least 25 minutes or 20-40 minutes, or 25-
35 minutes or
for about 30 minutes or for at least 35 minutes or at least 40 minutes or 30-
60 minutes or for
more than 60 minutes. Persons of ordinary skill in the art with resort to this
disclosure will
realize that pasteurization can also be accomplished at a higher temperature
in a shorter
period of time. Any suitable method of pasteurization can be used and examples
include vat
pasteurization, high temperature short time pasteurization (HTST), higher-heat
shorter time
(HEST) pasteurization, and in line pasteurization. Temperature and time
periods can be
selected accordingly.
[0037] When a pasteurization step is included it can be performed on the
biomass
subsequent to fermentation and prior to the acid wash step. In one embodiment
the steps can
include a pasteurization step, a homogenization step (e.g., bead milling), and
an acid wash
step, which may be performed in that order. In one embodiment the
pasteurization step is
performed prior to the homogenization step and/or prior to the acid wash step.
In one
embodiment the acid wash step is performed subsequent to the pasteurization
step. In
another embodiment the homogenization step is performed subsequent to the
pasteurization
step. The acid wash step can be performed either before or subsequent to the
homogenization
step and/or the pasteurization step. All of the steps can be performed in the
order recited and
additional steps can be performed before or after, or in between the recited
steps. In one
embodiment a solvent extraction (or solvent washing) step can be performed
subsequent to
the acid washing step.
[0038] A pasteurization step can be useful prior to the acid wash step when
the biomass
produces a protein with undesirable organoleptic properties, such as chytrids
and/or certain
types of algae often do. The pasteurization step can also occur prior to one
or more solvent
extraction step(s). Without wanting to be bound by any particular theory it is
believed that
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pasteurization destroys cellular lipases and/or other cellular enzymes and
prevents the
formation of some free fatty acids and oxidized lipids or other undesirable
compounds that
would lower the organoleptic quality of the product.
[0039]
These methods can yield a protein composition that has desirable organoleptic
properties, even if the biomass is comprised of organisms that produce a
proteinaceous
material or other materials that have undesirable organoleptic properties. The
methods can
convert the proteinaceous material derived from the biomass from one having
undesirable
organoleptic properties into a protein composition that has more desirable
organoleptic
properties, and one that is suitable or acceptable as a food or food
ingredient as measured by
performing acceptably in an organoleptic evaluation.
Delipidation and Solvent Washing
[0040] In some embodiments the methods involve one or more steps of mechanical
homogenization or mixing, which can involve (but is not limited to) bead
milling or other
high shear mixing (e.g. a ROTOSTAT mixer) or emulsifying. A homogenization
step can
involve the creation of an emulsion, a suspension, or a lyosol, and can
involve particle size
reduction and dispersion to provide smaller particles distributed more evenly
within a liquid
carrier. Homogenization produces a more uniform or "homogenized" composition,
such as a
more consistent particle size and/or viscosity of the mixture. A
batch or inline
homogenization step can be performed. A homogenization step can be performed
for at least
minutes or at least 10 minutes or at least 15 minutes or at least 20 minutes.
These one or
more steps can be followed by or separated by a step of centrifugation and
(optionally) re-
suspension in a buffer or solvent for an (optional) additional step of
homogenization or
mixing. Other mechanical stressors include, but are not limited to ultrasonic
homogenizers or
roto/stator homogenizers, or homogenizers that use high speed rotors or
impellers.
[0041] The
biomass can be subjected to one or more delipidation step(s). The one or more
delipidation step(s) can be done to the biomass prior to or after it is
subjected to an acid wash,
or both. Mechanic stress can be applied with the biomass in contact with an
appropriate
solvent. Thus, delipidation can involve a lipid extraction or solvent washing
step. A solvent
washing step involves exposure (or "washing") of the biomass to solvent for an
appropriate
period of time, which can be at least 5 minutes or at least 10 minutes or at
least 15 minutes or
about 15 minutes). The solvent can be any appropriate solvent, and in some
embodiments is
a polar solvent or a polar, protic solvent. Examples of useful polar, protic
solvents include,
but are not limited to ethanol, formic acid, n-butanol, isopropanol (IPA),
methanol, acetic
acid, nitromethane, hexane, acetone, water, and mixtures of any combination of
them. For
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example, in one embodiment the solvent can be a combination of hexane and
acetone (e.g.,
75% hexane and 25% acetone). In another embodiment the solvent in 90% or 100%
ethanol.
Any suitable ratio of solvent to biomass can be used such as, for example,
5:1, 6:1, 7:1, 8:1,
9:1, and other ratios. But the skilled person will realize other appropriate
solvents or
combinations that will find use in the invention. In various embodiments a
delipidation
and/or solvent washing/extraction step can involve a reduction in the lipid
content of the
mixture of at least 20% or at least 50% or at least 60% or at least 70% or at
least 80% or at
least 90% or at least 95% or at least 97% or at least 98%, all w/w.
[0042] The delipidation step can ensure proper lysing of the cells (e.g. by
mechanical
homogenization) comprising the biomass to maximize the protein extraction and
make lipidic
material available for extraction from the biomass. After mechanical
homogenization the
biomass can be separated by centrifugation and the lipidic materials in the
supernatant
removed. One or more additional steps of delipidation or solvent washing with
the solvent
can be performed to maximize delipidation. In some embodiments a second or
subsequent
cycle(s) of delipidation can utilize a different solvent than used in the
first cycle or in a
previous cycle to increase the chances of removing more undesirable compounds.
In some
embodiments a second solvent can also be included to provide for separation,
for example
including hexane and/or acetone or another hydrophobic solvent can provide for
separation
and thus extract more undesirable hydrophobic compounds. After homogenization
and at
least one solvent washing step (solvent washing can be done simultaneously
with
homogenization by homogenizing in the presence of solvent) the mixture or
biomass can be
referred to as a delipidated biomass. The biomass can also have been subjected
to
mechanical homogenization as a separate step before the solvent washing steps.
[0043] Without wishing to be bound by any particular theory it is believed
that compounds
having undesirable organoleptic taste and smell properties are removed or
inactivated in the
pasteurization step and/or the one or more delipidation or solvent washing
step(s) and/or the
one or more acid wash step(s) and/or the one or more steps of solvent washing
that can be
applied following the one or more acid washing step(s). Additional substances
with
undesirable organoleptic properties can be removed by repeating any of the
steps one or two
or three or more than three times. Additional processes described herein can
also be
performed as one or more steps in the methods of making or synthesizing a
protein material.
The result of the processes is a material that is high in protein content and
derived from
biomass.
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[0044] In various embodiments the protein material prepared according to
the invention
has a reduced lipid content. In some embodiments the methods of the invention
reduce the
lipid content of the biomass from more than 20% or more than 15% or more than
10% or
more than 8% or more than 7% or more than 6% or more to 5% to less than 20% or
less than
18% or less than 15% or less than 13% or less than 10% or less than 7% or less
than 5% lipid
content or less than 4% lipid content or less than 3% or less than 2% lipid
content or less than
1% lipid content or less than 0.75% lipid or less than 0.6% lipid or less than
0.5% lipid, all
w/w, present in the protein product material.
Acid Wash
[0045] In some embodiments the biomass is subjected to one or more acid
wash step(s).
The acid wash step can be performed on pasteurized and/or delipidated biomass.
Acid
washing can comprise exposing the delipidated biomass to acid or a depressed
pH for a
period of time. The biomass, and therefore the proto-protein it contains, can
be exposed to
the acid wash in a solution, suspension, slurry, or any suitable state. The
acid wash can
utilize any suitable inorganic acid or a suitable organic acid. The inorganic
acids are derived
from one or more inorganic compounds that form hydrogen ions when dissolved in
water.
Examples include, but are not limited to, sulfuric acid, nitric acid,
phosphoric acid, boric acid,
hydrochloric acid, hydrofluoric acid, hydrobromic acid, and perchloric acid.
The person of
ordinary skill will realize other inorganic acids that also function in the
invention. The
delipidated biomass can be mixed with water to generate an aqueous mixture.
The acid
solution (e.g., 1M sulfuric acid) can then be pipetted into the mixture until
the pH is reduced
to a depressed pH. In various embodiments the pH can be adjusted to a
depressed pH of
about 4.0 or about 3.8 or about 3.5 or about 3.3 or about 3.2 or about 3.0 or
about 2.8 or about
2.5 or from about 2.0 to about 2.5 or from about 2.0 to about 3.0, or from
about 2.0 to about
4.0, or from about 2.0 to about 3.5, or from about 2.2 to about 2.8, or from
about 2.3 to about
2.7, or from about 2.2 to about 3.8, or from about 2.3 to about 3.7, or from
about 2.5 to about
3.0, or from about 2.8 to about 3.2, or from about 3.0 to about 3.5, or from
about 3.2 to about
3.8. In other embodiments the pH can be adjusted to less than about pH 4.0 or
less than about
pH 3.7 or less than about pH 3.6 or less than about pH 3.5 or less than about
pH 3.3 or less
than about pH 3.0 or less than about pH 2.7 or less than about pH 2.5. The
mixture can then
be held at the indicated pH for a period of time. The mixture can also be
mixed or stirred or
incubated for the period of time, or a portion thereof. The period of time can
be any of at
least 1 minute or at least 5 minutes or at least 10 minutes or at least 20
min. or at least 30 min,
or from about 20 minutes or about 30 minutes, or about 40 minutes, or from 10-
30 minutes,
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or from 10-40 minutes, or from 20-40 minutes, or from 20 minutes to 1 hour, or
from 10
minutes to 90 minutes, or from 15 minutes to 45 minutes, or at least 1 hour or
about 1 hour or
at least 90 minutes or at least 2 hours.
[0046] After the biomass has been exposed to the depressed pH for an
appropriate period
of time (and optional mixing) the pH can then be raised to a raised pH by
addition of a basic
or alkaline compound, for example KOH. Persons of ordinary skill in the art
will realize that
other basic or alkaline compounds can also be used, for example sodium
hydroxide, calcium
hydroxide, or other basic compounds. The basic compound can be added at any
convenient
concentration, e.g., about 1 M or 0.5-1.5 M or 0.75-1.25M. The basic compound
can be
added until the pH is adjusted to a raised pH of about 4.5. But in other
embodiments the
raised pH can be about 4.0 or about 4.2 or about 4.7 or about 5Ø In more
embodiments the
pH can be raised to greater than 4.0 or greater than 4.2 or greater than 4.5
or greater than 4.7
or greater than 5Ø After the pH adjustment to the raised pH the mixture can
be stirred or
incubated for an appropriate period of time, which in some embodiments is
about 30 min or
about 1 hour or about 90 minutes or more than 30 minutes or more than 1 hour.
[0047] When the pH is adjusted to the depressed pH there is a noticeable
decrease in the
viscosity of the mixture from a thick slurry of poor mixing capability to a
thin, watery
consistency of markedly lower viscosity (i.e. there is an observable decrease
in viscosity).
The decrease in viscosity can be observed at the start of the acid addition
by, for example, the
inability of a common laboratory overhead mixer to be able to fully blend the
solution
(cavitation at the impeller). As the pH is lowered the change in viscosity can
be observed as
changing to a viscosity similar to a watery solution requiring a reduction in
the impeller
tipspeed to avoid splashing of the solution. Thus, the change in viscosity can
be a decrease of
at least 10% or at least 20% or at least 30% or at least 40% or at least 50%,
as measured by
standard methods of measuring viscosity such as a viscometer. Examples of
methods of
measuring viscosity include, but are not limited to, a glass capillary
viscometer or a vibrating
needle viscometer, a rheometer, a rotational rheometer, and the inclined plane
test, but any
suitable method can be utilized. When the pH is adjusted upwards to the raised
pH the
viscosity of the mixture increases, but does not achieve its viscosity prior
to exposure to
acidic conditions, revealing that a marked, irreversible, and permanent
chemical change has
occurred from the initial protein-containing mixture derived from the biomass.
[0048] Without wanting to be bound by any particular theory it is believed
that subjecting
the proto-protein to the delipidation and/or acid wash and/or other processes
described herein
may free or dissociate bound lipids by making (possibly irreversible)
conformational changes
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in the proto-protein. It may also result in cleavage of covalently bound lipid-
protein
conjugates. The acid wash step does not truly hydrolyze the proteins in the
biomass, but
rather frees lipid moieties from the proteinacious (proto-protein) molecules
in the biomass.
The step may cause a conformational change in the proteins, and thereby
freeing the lipidic
moieties and allowing them to be removed. These processes may make the lipid
species (or
other solvent soluble molecules) available for removal during solvent washing
and/or
extraction steps. These steps, and possibly in combination with the additional
steps described
herein, are believed to thus remove the portions of the proto-protein that
give the undesirable
organoleptic properties, and thus provide the organoleptically acceptable
protein material that
is the food or food ingredient of nutritional interest in the invention, which
is thus harvested.
The protein-containing food or food product produced by the processes
described herein is
thus a markedly different molecule than the proto-protein that begins the
processes.
Post-Acid Wash Re-washing (re-working) steps
[0049] Following the acid wash step there can be one or more steps of
solvent extraction
or washing, each optionally followed by a step of centrifugation to achieve a
pellet, and
resuspension in a solvent. The solvent can be any appropriate solvent as
described herein for
a solvent washing and/or delipidation step. After these steps, (if performed)
post acid wash,
the protein mixture can be optionally dried in a rotary evaporator to make a
protein
concentrate, which can be utilized as a food or food ingredient.
Proto-Protein
[0050] In some embodiments the biomass contains a proto-protein, which is a
protein-
containing molecule which also contains a significant non-protein moiety. In
some
embodiments the non-protein moiety is a lipid moiety. The proto-protein can be
the protein
produced by the biomass from which it is derived in its natural form, and
before being treated
according to the methods described herein. In some embodiments the proto-
protein is close
to the natural form proteinaceous material and has undesirable or unfavorable
organoleptic
taste and smell properties and would score low on the "degree of liking" scale
or other
method of evaluating organoleptic properties. Various algae and microbes
produce proteins
with these characteristics, and in some embodiments the proto-protein is an
algal protein with
undesirable organoleptic properties. In the methods of the invention the
biomass can be
converted into a protein-containing food or food ingredient that has more
desirable
organoleptic properties and scores as acceptable on methods of evaluating such
properties.
Without wanting to be bound by any particular theory it is believed that the
proto-protein
may contain a lipidic component that gives the undesirable organoleptic taste
and/or smell
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properties. Removal or disrupting of this protein (or its lipidic component)
results in an
improvement in organoleptic properties. In addition to (or instead of) lipid
moieties the
proto-protein can have other, molecular components or moieties that cause it
to have (or
worsen) its undesirable organoleptic properties. Therefore by applying the
methods
described herein the protein component of the biomass is converted into an
organoleptically
acceptable protein composition of the invention.
[0051] The molecular weight distribution of the proto-protein refers to the
percentage of
proto-protein molecules having a molecular weight within a specified size
range or ranges.
For example, the proto-protein may have a molecular weight distribution so
that at least 50%
or at least 60% or at least 70% or at least 80% of the proto-protein molecules
(by weight)
have a molecular weight of between about 10,000 and about 100,000 daltons, or
from about
10,000 to about 50,000 daltons, or from about 20,000 to about 100,000 daltons,
or from about
20,000 to about 80,000 daltons, or from about 20,000 to about 60,000 daltons,
or from about
30,000 to about 50,000 daltons, or from about 30,000 to about 70,000 daltons,
all non
aggregated. In other embodiments at least 70% or at least 80% of the proto-
protein
molecules have a molecular weight of between about 10,000 and about 100,000
daltons, or
from about 20,000 to about 80,000 daltons, or from about 30,000 to about
50,000 daltons, or
from about 30,000 to about 70,000 daltons, all non-aggregated. In other
embodiments the
molecular weight distribution of the proto-protein may be such that less than
25% or less than
10% or less than 5% of the proto-protein molecules have a molecular weight
below about
20,000 daltons or below about 15,000 daltons or below about 10,000 daltons.
[0052] The methods of the invention convert a biomass containing a proto-
protein into a
proteinaceous or protein-rich concentrate. The fatty acid methyl ester (FAME)
profile of the
biomass at various steps can be evaluated to determine the quantity of lipidic
material
removed during the processes. Table 1 and Figure 1 show the percent removal of
FAME by
the processing steps of the invention.
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Table 1 ¨ Percent removal of FAME by processing steps
Sample ID Process Step
First Bead Second Bead Acid Wash Final
Milling Milling
505-002 25% 26% 59%
506-002 19% 34% 21% 79%
514-002 8% 50% 24% 80%
average 13.5% 33% 24%
[0053] The values in Table 1 reflect the percent of lipid removed by the
indicated process
step from the input material at that step. "Final" indicates the percent of
total lipid removed
versus the lipid content of the starting biomass. The data corresponds to the
graph in Figure
3. In various embodiments at least 60% or at least 70% or at least 75% of the
lipid content in
the fermented biomass that begins the methods is removed by the methods of the
invention.
[0054] In some embodiments the biomass (or proto-protein) has a % FAME of
greater
than 9% or greater than 10% or greater than 11% or greater than 12% or greater
than 13%.
As a result of the methods described herein the % FAME can be reduced to less
than 5% or
less than 4% or less than 3% or less than 2% or less than 1% or less than
0.75% or less than
0.50%, all w/w.
[0055] The para-anisidine test (pAV), which is a standard test for
secondary oxidation
products of lipids, can also be used to monitor the amount of secondary
oxidation products of
lipids present after the processes of the invention, and therefore further
characterize the
protein product produced by the methods of the invention. In some embodiments
the protein
product produced by the methods of the invention has a pAV value of less than
2.0 or less
than 1.0 or less than 0.9 or less than 0.8 or less than 0.7 or less than 0.6
or less than 0.5.
Additional Methods
[0056] In some embodiments the invention provides methods of increasing the
protein
content of a biomass. In some embodiments the product of the invention is a
protein-
containing product having a higher protein concentration than the original
biomass, with
neutral color and improved hedonic or organoleptic properties. In various
embodiments the
protein-containing biomass that enters the processes of the invention can have
a protein
content of less than 65% or 50-65% or 40-70% or 45-65% or 45-70% (all w/w) and
the
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protein content of the product of the methods is raised to greater than 65% or
greater than
680 o or greater than 70% or greater than 72% or greater than 750 o or greater
than 770 o or
greater than 80% or 70-90% or 65-90% or 70-90% or 72-87% or 75-85% or 75-80%.
[0057] The invention also provides methods of lowering the arginine and
glutamic acid
(or glutamic acid and glutamine) content of a protein material. Arginine and
glutamic acid
(and glutamine) are amino acids that are generally easy to find in various
types of food
products. In many embodiments it is desirable to have a protein-rich food or
food ingredient
that has a lower content of these common amino acids and a more balanced
supply of the 20
essential amino acids. The methods of the invention produce a protein product
or food
ingredient with a lower amount of glutamic acid (or glutamic acid and
glutamine) and
arginine than is commonly available in food sources. In various embodiments
the percent of
glutamic acid (or glutamic acid and glutamine) is lowered from more than 21%
or more than
22 A to less than 20% or less than 19 A (% of total amino acids). The percent
of arginine is
lowered from more than 9 A to less than 9 A (% of total amino acids). The
methods of
lowering the arginine and glutamic acid (or glutamic acid and glutamine)
content comprise
any of the methods described herein.
Example 1
[0058] This example provides a general scheme for producing a dried protein
material or
concentrate (e.g., a powder) containing a proto-protein from algal biomass.
This example
illustrates a specific method but persons of ordinary skill with resort to
this disclosure will
realize other embodiments of the methods, as well as that one or more of the
steps included
herein can be eliminated and/or repeated. Furthermore, any of the steps
described herein can
be included in any of the methods.
[0059] In this example algae (chytrids) of the genus Aurantiochytrium sp.
were used and
were cultivated in a fermenter containing a marine medium containing 0.1 M
glucose and 10
g/L of yeast extract (or peptone substitute), which supplied a source of
organic carbon. The
medium also contained macronutrients, including 0.1M NaC1, 0.01M CaC12, 0.04M
Na2SO4,
0.03M KH2PO4, 0.04M (NH4)2SO4, 0.006M KC1, 0.02M MgSO4), plus nanomolar
quantities
of vitamin B12, thiamine and biotin. The culture was maintained at 30 C for
24 hours with
300-80 rpm of agitation, 0.1 vvm to 1.0 vvm aeration, 50% dissolved oxygen,
and pH
controlled to 6.3 0.1 using 30 A NaOH.
[0060] After harvesting, the fermentation broth was removed from the cells
via
centrifugation and the resulting biomass pellet was diluted in water and re-
centrifuged (cell
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wash). The resulting paste was mixed with antioxidants to prevent oxidation of
oils and other
components, and then drum dried to remove water, which produced a dry cellular
material.
[0061] A pasteurization step was performed by raising the temperature of
the broth to
about 65 C and holding it at that temperature for about 30 minutes. The dry
cells were then
thoroughly lysed in 100% ethanol in a bead mill. This is a homogenization and
solvent
extraction step and removes soluble substances such as lipids, and the
delipidated biomass is
separated from the miscella using centrifugation.
[0062] The biomass was then subjected to an acid wash via titration of 1 N
H2SO4, until
the pH was acidified to about 3.5. The biomass was then mixed for about 30
minutes. The
pH was then raised to about 4.5 with 1 N NaOH and the biomass mixed for 1
hour.
[0063] The acid washed material was then centrifuged and the supernatant
removed. The
pellet was then subjected to two re-washing/extraction steps, which involved
two rounds of
suspension in 100% ethanol followed by high shear mixing and centrifugation.
The
supernatant was decanted to maximize extraction and removal of undesired
compounds. The
high shear mixing was performed with a rotor stator type mixer (e.g., IKA
ULTRA-
TURRAXg) with the temperature being controlled at < 20 C by an ice bath. The
resultant
ethanol-washed pellet (biomass) was then dried by placing in a modified rotary
evaporation
flask to promote tumble-drying at room temperature under moderate vacuum.
After
approximately 4 hours the material changed from a paste to a powder. At this
point, the
material was removed from the rotary evaporator and ground to a fine powder
with a mortar
and pestle. This material was then placed on an aluminum tray in a vacuum oven
at 90 C for
approximately 11 hours to remove any residual solvent or moisture. Once dry,
the material
was passed through a particle size classifier to remove particles greater than
300 um in size.
These particles can be completely removed from the final product if desired,
or further
ground up and returned back to the final product. The end result of the
process was a
uniform, neutral colored powder of neutral hedonic character, which can be
packaged under
nitrogen and stored in a -80 C freezer.
Example 2
[0064] Three independent fermentations were performed on algae of the genus
Aurantiochyrium sp. in medium similar to that of Example 1 and the mass of the
acid wash
supernatant stream was quantitated, and protein determined by the Dumas method
(quantitative determination of Nitrogen by elemental analysis). As shown in
Table 2 below,
the acid wash removed between 8.8% and 15.8% of the initial feedstock mass.
Converting
nitrogen content to protein content by the calculation (N * 6.25) estimates
the protein content
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of the acid wash solids is 12.15% to 15.50% protein. The protein removed by
the acid wash
step ranged from 2.01% to 3.4% of the initial protein in the feed.
Table 2 ¨ Acid Wash Supernatant Masses and Protein
Sample 825 Sample 908 Sample 319
Mass
removed, % 15.80% 14.00% 8.80%
of feed
Acid wash
Solids % 12.60% 12.15% 15.50%
protein
Protein, % of
3.40% 2.70% 2.01%
feed Protein
Example 3
[0065] An additional example of the impact of the acid wash upon amino acid
composition is shown below. Two separate processes were performed where the
acid wash
supernatant was dialyzed and dried, and analyzed for amino acid composition.
An
Aurantiochytrium (chytrid) strain was processed as described above, the acid
wash
supernatant and algal protein concentrate were analyzed and compared to the
initial dry
biomass feed. It was found that glutamic acid (or glutamic acid and glutamine)
and arginine
are selectively removed from the biomass during the acid wash.
[0066] Without wanting to be bound by any particular theory it is believed
that the acid
wash step prepares the proteinaceous material for a preferential protein
removal so that the
content of generally unwanted amino acids arginine, glutamic acid (or glutamic
acid and
glutamine), and hydroxyproline is lowered in the final protein product versus
the raw algal
protein. After acid washing the samples were subjected to two additional
rounds of solvent
washing. It is also believed that the acid wash step exposes or otherwise
renders certain
proteins in the proteinaceous material susceptible to removal, and these
removed proteins are
high in the content of these unwanted amino acids. This is advantageous since
it allows for
the production of a more nutritionally balance protein material. The content
of arginine and
glutamic acid (or glutamic acid and glutamine) and hydroxyproline is measured
by
calculating the ratio of each amino acid in the final protein product pellet
versus the content
in the supernatant. Thus a low ratio indicates the amino acid is more
prevalent in the
supernatant. Table 3 below illustrates the data and shows that the ratio for
these three amino
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acids is less than 2 or less than 1 or less than 0.75 for arginine, less than
2 or less than 1 or
less than 0.75 or less than 0.60 for glutamic acid (or glutamic acid and
glutamine), and less
than 2 or less than 1 or less than 0.75 or less than 0.55 for hydroxyproline.
Table 3
Ratio of Pellet
Acid Wash Final
Amino Acid % to AWS
Supernatant Product in
of sample amino acid
(AWS) Pellet
composition
Methionine 0.08% 0.83% 10.35
Cystine 0.13% 0.48% 3.80
Lysine 0.76% 4.38% 5.76
Phenyl al anine 0.01% 2.82% 315.04
Leucine 0.21% 4.56% 21.26
Isoleucine 0.19% 2.33% 12.40
Threonine 0.50% 3.07% 6.13
Valine 0.33% 3.66% 11.07
Hi sti dine 0.35% 1.76% 5.04
Arginine 15.61% 11.12% 0.71
Glycine 0.95% 3.23% 3.40
Aspartic Acid 1.17% 6.86% 5.86
Serine 0.57% 3.27% 5.71
Glutamic Acid 76.24% 41.97% 0.55
Proline 0.35% 2.64% 7.58
Hydroxyproline 0.05% 0.03% 0.49
Alanine 1.70% 4.20% 2.48
Tyrosine 0.72% 2.27% 3.18
Tryptophan 0.09% 0.79% 8.87
TOTAL: 100.00% 100.00% 1.00
Example 4 - Lipid Removal During Acid Wash
[0067] Two processes using the same biomass source (chytrid #705) were
performed to
show the effect of the acid wash on FAME content in the protein concentrate.
After drum
drying the initial biomass from the fermenter the samples were subjected to
two rounds of
mechanical homogenization by bead milling followed by a step of solvent
washing in 100%
isopropyl alcohol. Sample 225-002/A was subjected to an acid washing step as
describe in
Example 1 while sample 225-002/A.2 was not. Each sample was then subjected to
two
reworking solvent washing steps in 100% isopropyl alcohol before being dried
in a rotary
evaporator. The results clearly show the lowering of the final FAME content in
the protein
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product from 2.19% of final dry weight to 0.89% of final dry weight, which can
be
attributable to the acid washing step.
Table 4
Protein
Experimental %
Protein concentrate
Lot Designation Sample Descriptor
Descriptor
(Dumas) FAME% of
dry weight
Drum Dry / IPA Mill / AW /
225-002/A Acid Washed 83.66%
0.89%
Rework / Drying
Drum Dry/ IPA Mill /
225-002/A.2 Non-Acid Washed Rework / Drying (No acid 81.22%
2.19%
wash)
[0068] The
stepwise efficiency of removing available lipids through the process was
examined in order to see the specific contribution of the acid wash step for
the removal of
lipids. Figure 1 shows the results for three independent treatments performed
using the strain
from Example 3. Ethanol was used as the solvent prior to and after the acid
wash. The acid
wash step included a first adjustment to pH 3.5 with 1 N H2504 per Example 1,
followed by
adjustment to pH 4.5 with 1 N KOH. For each significant process step, the
resultant solids
were analyzed for FAME content and a percent of available FAME that was
removed in the
step was calculated, as shown in Figure 1. The acid wash step removed 26%,
21%, and 24%
of the lipid present in the biomass after the bead mill processing (samples
505-002, 506-002,
and 514-002, respectively). The data show that when an acid wash step is
included in the
preparation method the percent of FAME in the protein produce produced is
reduced 0.89%,
or to less than 1%. When the acid wash step is omitted from the process the
percent FAME
in the protein produce is 2.19%, or higher than 2%.
Example 5
[0069] The
para-anisidine test (pAV), which is a standard test for secondary oxidation
products of lipids, was used to monitor the amount of secondary oxidation
products of lipids
present after certain steps of the methods. The pAV values were determined for
four
independently-fermented batches of chytrid biomass, tested at three steps in
the downstream
processing: water-washed biomass collected immediately at the conclusion of
fermentation
(washed pellet); pasteurized biomass; final protein concentrate (after acid
washing and two
re-working steps). The downstream process steps are shown in the process flow
diagram of
Figure lb and described in Table 5 below.
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Table 5 ¨ pAV Relative to Soy Protein
p-AV relatice to Washed Pasteurized Protein
soy protein Pell et Biomass Concentrate
IP-150505-002 4.0 4.0 0.8
IP-150506-002 3.6 5.4 0.5
IP-150511-002 3.5 2.5 0.8
IP-150514-002 1.6 1.5 0.4
[0070] The values shown in Table 5 are ratios of the pAV of the algal
protein concentrate
relative to the pAV value determined for a commercially available protein
isolate produced
from soybean (which is used as a benchmark standard). The data show that prior
to the
processing steps of bead milling/ethanol extraction and acid washing, the
algal protein
concentrate has a higher content of secondary lipid oxidation products than
does a soybean
protein isolate. But after two bead milling/ethanol solvent washing steps and
one acid
washing step with two reworking solvent washing steps, each of the four
samples of protein
product have a lower content of secondary lipid oxidation products than the
soybean protein
isolate. Thus, the steps of the invention, including the acid washing, improve
the quality of
the protein concentrate with respect to lipid content (and therefore lipid
oxidation) and
organoleptic properties.
Example 6 ¨ Sensory Panels
[0071] Reports from sensory panels composed of persons selected to evaluate
the
organoleptic properties of the protein composition have demonstrated the
processes of the
present invention result in improved organoleptic (hedonic) properties. The
presence of an
unpleasant fishy odor or taste, or ammonia-like odor or taste, or briny odor
or taste was
markedly decreased as a result of the process while the protein material
maintained a high
protein content.
[0072] Persons of ordinary skill in the art understand how to assemble a
sensory
evaluation panel and evaluate food samples in a reliable manner, for example
the 9 point
hedonic scale, which is also known as the "degree of liking" scale can be
utilized. (Peryam
and Girardot, N.F., Food Engineering, 24, 58-61, 194 (1952); Jones et al. Food
Research, 20,
512-520 (1955)). This example therefore provides only one scientifically valid
manner of
performing such evaluation.
[0073] A panel of six adult subjects (3 male and 3 female) evaluate the
organoleptic taste
and smell properties of eight protein products derived from algal (chytrid)
biomass processed
as described in Example 1. The subjects are randomly assigned an identifying
letter A-F.
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Four of the eight samples are prepared according to the procedure of Example
1, which
includes one acid wash procedure ("test" samples). The other four samples are
control
samples, which have been prepared identically except they were not subjected
to the acid
washing step ("control" samples). After the samples are dried and obtained in
powdered
form, 1 gram of protein powder is dissolved in deionized water to make a 10%
solution in a
plastic tube. The eight samples are provided to each subject in random order
and without any
subject knowing the identity of any sample.
[0074] The samples are evaluated for whether the samples are
organoleptically pleasing
or unpleasant. The subjects are asked to consider the categories "fishy taste
and/or smell"
and "ammonia-like taste and/or smell" and "briny taste and/or smell" according
to the
following five point scale: 0 ¨ none; 1 ¨ slight; 2 ¨ moderate; 3 high; and 4
¨ extreme. The
subjects also evaluate the general organoleptic properties as acceptable or
unacceptable, using
soy protein similarly prepared as a standard, and whether the samples have
organoleptic
properties equal to, better, or worse than the soy protein sample. The
subjects are instructed
to assign the sample the lowest rating received in either category. The manner
of testing is
first to evaluate the aroma of the sample. If the subject rates the aroma a 3
or 4 in any
category the sample is considered organoleptically unpleasant or unacceptable
and no tasting
is required. If the aroma rates between 0 and 2 the subject further tests the
sample by the
known "sip and spit" method, with sample being held in the mouth for 1-2
seconds.
[0075] In the aroma evaluation portion of the study, 5 of the 6 panel
members rate all
four control samples a 3, i.e., high fishy smell and/or high ammonia-like
smell and/or high
briny smell, and therefore organoleptically unacceptable. The subjects also
rate the control
samples as less pleasing than the soy protein sample. Therefore these 5
subjects do not
proceed to the taste portion of the study for these samples and the samples
are rated as having
unpleasant or unacceptable organoleptic properties. The remaining subject
rates three of the
four control samples a "3", and the remaining control sample a "2." For the
fourth control
sample this subject proceeds to the taste portion and rates the remaining
control sample a 3
and rates all samples less pleasing than the soy sample.
[0076] For the four test samples in the aroma portion of the study, 5 of
the 6 subjects rate
all four of the samples a "0" and equal to soy. The remaining subject rates
three samples a
"0" and equal to soy and one sample a 1 and less pleasing than soy.
[0077] The subjects then proceed to the taste portion of the study. For the
taste portion
five of the subjects rate all four samples a "0" for taste and equal to soy.
The remaining
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subject rates three samples a "0" and equal to soy, and one sample a "1" and
less pleasing
than soy.
[0078] The data are summarized in Table 6 and show that the protein
composition
prepared according to the present invention has improved organoleptic
properties versus
samples prepared according to traditional methods. It is also seen that
samples prepared
according to the invention are clearly more likely to be equal to soy protein
standard in
organoleptic taste and smell properties and to have acceptable or desirable
organoleptic
properties.
Table 6 ¨ Samples Evaluated as either organoleptically pleasing or unpleasant
A B C D E F
S ¨ 0 S ¨ 0 S - 0 S ¨ 0 S ¨ 0 S ¨ 0
1 test
T ¨ 0 T ¨ 0 T ¨ 0 T - 0 T - 0 T ¨ 0
S ¨ 0 S ¨ 0 S - 0 S ¨ 1 S ¨ 0 S ¨ 0
2 test
T ¨ 0 T ¨ 0 T - 0 T ¨ 1 T - 0 T ¨ 0
S ¨ 0 S ¨ 0 S - 0 S ¨ 0 S ¨ 0 S ¨ 0
3 test
T ¨ 0 T ¨ 0 T ¨ 0 T ¨ 0 T ¨ 0 T ¨ 0
S ¨ 0 S ¨ 0 S-0 S ¨ 0 S ¨ 0 S ¨ 0
4 test
T ¨ 0 T - 0 T - 0 T ¨ 0 T - 0 T - 0
5 control S ¨ 3 S ¨ 3 S-3 S ¨ 3 S ¨ 3 S ¨ 3
S ¨ 2
6 control S ¨ 3 S-3 S ¨ 3 S ¨ 3 S ¨ 3
T ¨ 3
7 control S ¨ 3 S ¨ 3 S-3 S ¨ 3 S ¨ 3 S ¨ 3
8 control S ¨ 3 S ¨ 3 S-3 S ¨ 3 S ¨ 3 S ¨ 3
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