U.S. patent application number 13/941211 was filed with the patent office on 2014-08-07 for method and compositions for consumables.
This patent application is currently assigned to MARAXI, INC.. The applicant listed for this patent is MARAXI, INC.. Invention is credited to Patrick Brown, Michael Eisen, Sergey Solomatin, Ranjani Varadan, Marija Vrljic.
Application Number | 20140220217 13/941211 |
Document ID | / |
Family ID | 51259423 |
Filed Date | 2014-08-07 |
United States Patent
Application |
20140220217 |
Kind Code |
A1 |
Brown; Patrick ; et
al. |
August 7, 2014 |
METHOD AND COMPOSITIONS FOR CONSUMABLES
Abstract
Disclosed herein are improved methods and compositions which
more accurately replicate the characteristics that consumers value
in the preparation and consumption of meat and which overcome the
shortcomings and drawbacks of current meat substitutes.
Inventors: |
Brown; Patrick; (Stanford,
CA) ; Vrljic; Marija; (San Mateo, CA) ;
Varadan; Ranjani; (San Jose, CA) ; Eisen;
Michael; (Berkeley, CA) ; Solomatin; Sergey;
(Mountain View, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
MARAXI, INC. |
Menlo Park |
CA |
US |
|
|
Assignee: |
MARAXI, INC.
Menlo Park
CA
|
Family ID: |
51259423 |
Appl. No.: |
13/941211 |
Filed: |
July 12, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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PCT/US2012/046560 |
Jul 12, 2012 |
|
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13941211 |
|
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61671069 |
Jul 12, 2012 |
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61572205 |
Jul 12, 2011 |
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Current U.S.
Class: |
426/574 |
Current CPC
Class: |
A23L 13/424 20160801;
A23J 1/12 20130101; A23J 3/227 20130101; A23L 27/26 20160801; A23L
27/00 20160801; A23L 27/20 20160801; A23J 1/14 20130101; A23J 3/14
20130101; A23L 13/426 20160801; A23L 27/10 20160801; A23L 33/185
20160801 |
Class at
Publication: |
426/574 |
International
Class: |
A23J 1/00 20060101
A23J001/00; A23J 1/14 20060101 A23J001/14; A23J 1/12 20060101
A23J001/12; A23J 1/06 20060101 A23J001/06 |
Claims
1.-162. (canceled)
163. A meat substitute product, comprising: a) a muscle replica; b)
a fat tissue replica; and c) a connective tissue replica; wherein
said muscle replica, fat tissue replica, and/or connective tissue
replica are assembled in a manner that approximates the physical
organization of meat.
164. The meat substitute product of claim 163, comprising two or
more of said muscle replica, fat tissue replica, and connective
tissue replica.
165. The meat substitute product of claim 163, wherein (i) said
muscle replica accounts for 40-90% of said product by weight, (ii)
said fat tissue replica accounts for 1-60% of said product by
weight, and (iii) said connective tissue replica accounts for 1-30%
of said product by weight.
166. The meat substitute product of claim 163, comprising: a)
60-90% water; b) 5-30% protein content; c) 1-20% of a fat or fat
replica; wherein said protein content comprises one or more
isolated, purified plant proteins.
167. The meat substitute product of claim 166, wherein said protein
content is derived from a non-animal source.
168. The meat substitute product of claim 167, wherein said
non-animal source is a plant source, or a genetically modified
yeast or bacteria.
169. (canceled)
170. The meat substitute product of claim 163, wherein 50% or more
of said protein content by weight are one or more isolated purified
proteins.
171. The meat substitute product of claim 170, wherein each of said
one or more isolated proteins is isolated and purified separately
from different plant species.
172. The meat substitute product of claim 170, wherein one or more
of said isolated proteins is selected from the group consisting of:
hemoglobin, myoglobin, chlorocruorin, erythrocruorin, neuroglobin,
cytoglobin, protoglobin, truncated 2/2 globin, HbN, cyanoglobin,
HbO, Glb3, and cytochromes, Hell's gate globin I, bacterial
hemoglobins, ciliate myoglobins, flavohemoglobins, ribosomal
proteins, actin, hexokinase, lactate dehydrogenase, fructose
bisphosphate aldolase, phosphofructokinases, triose phosphate
isomerases, phosphoglycerate kinases, phosphoglycerate mutases,
enolases, pyruvate kinases, glyceraldehyde-3-phosphate
dehydrogenases, pyruvate decarboxylases, actins, translation
elongation factors, ribulose-1,5-bisphosphate carboxylase oxygenase
(rubisco), ribulose-1,5-bisphosphate carboxylase oxygenase activase
(rubisco activase), albumins, glycinins, conglycinins, globulins,
vicilins, conalbumin, gliadin, glutelin, gluten, glutenin, hordein,
prolamin, phaseolin (protein), proteinoplast, secalin, extensins,
triticeae gluten, zein, any seed storage protein, oleosins,
caloleosins, steroleosins or other oil body proteins, vegetative
storage protein A, vegetative storage protein B, moong seed storage
8S globulin.
173. The meat substitute product of claim 163, further comprising
one or more isolated and purified iron-containing proteins.
174. The meat substitute product of claim 173, wherein said one or
more isolated and purified iron-containing proteins is selected
from the group consisting of hemoglobin, myoglobin, leghemoglobin,
non-symbiotic hemoglobin, chlorocruorin, erythrocruorin,
neuroglobin, cytoglobin, protoglobin, truncated 2/2 globin, HbN,
cyanoglobin, HbO, Glb3, Hell's gate globin I, bacterial
hemoglobins, ciliate myoglobins, and flavohemoglobins.
175. The meat substitute composition of claim 174 wherein said
iron-containing protein comprises an amino acid sequence with at
least 70% homology to SEQ ID NO 1.
176. The meat substitute product of claim 163, wherein said meat
substitute product contains no methylcellulose, no carrageenan, no
caramel color, no konjac flour, no gum arabic, and no acacia
gum.
177. The meat substitute product of claim 174, wherein said meat
substitute product contains less than 1% wheat gluten.
178. The meat substitute product of claim 175, wherein said meat
substitute product contains no wheat gluten.
179. The meat substitute product of claim 176, wherein said meat
substitute product is characterized by one or more of the following
contains no soy protein isolate, no soy protein concentrate, or no
tofu.
180. (canceled)
181. (canceled)
182. The meat substitute product of claim 179, wherein said meat
substitute product contains less than 5% carbohydrates.
183. (canceled)
184. The meat substitute product of claim 163, wherein said meat
substitute product is characterized by one or more of the
following: contains no tofu, no soy protein, contains less than 1%
cellulose, contains less than 5% insoluble carbohydrates, or no
wheat gluten.
185. (canceled)
186. The meat substitute product of claim 163, wherein said meat
substitute product contains no animal products and less than 5%
carbohydrates.
187. (canceled)
188. (canceled)
189. (canceled)
190. (canceled)
191. (canceled)
192. The meat substitute product of claim 163, wherein said meat
substitute product contains no wheat gluten, and less than 5%
insoluble carbohydrates.
193-222. (canceled)
Description
CROSS REFERENCE
[0001] This application is a continuation-in-part of International
Application No. PCT/US2012/046560, filed Jul. 12, 2012, which
claims the benefit of priority of U.S. Provisional Application
61/572,205, filed Jul. 12, 2011 and is a non-provisional of and
claims the benefit of U.S. Provisional Application 61/671,069,
filed Jul. 12, 2012, all of which are incorporated herein by
reference.
BACKGROUND OF THE INVENTION
[0002] Animal farming has a profound negative environmental impact.
Currently it is estimated that 30% of Earth's land surface is
dedicated to animal farming and that livestock account for 20% of
total terrestrial animal biomass. Due to this massive scale animal
farming accounts for more than 18% of net greenhouse gas emissions.
Animal farming may be the largest human source of water pollution,
and animal farming is by far the world's largest threat to
biodiversity. It has been estimated that if the worlds human
population could shift from a meat containing diet to a diet free
of animal products, 26% of Earth's land surface would be freed for
other uses. Furthermore the shift to a vegetarian diet would
massively reduce water and energy consumption.
[0003] The consumption of meat has a profound negative impact on
human health. The heath benefits of a vegetarian diet are well
established. If the human population would shift to a vegetarian
diet the cost savings in health care would be significant.
[0004] Hunger is a worldwide problem, yet the world's 4 major
commodity crops (soybeans, maize, wheat, and rice) already supply
more than 100% of the human population's requirements for calories
and protein, including every essential amino acid.
[0005] Plant based meat substitutes have largely failed to cause a
shift to a vegetarian diet. The current state of the art for meat
substitute compositions involves the extrusion of soy/grain
mixture, resulting in products which largely fail to replicate the
experience of cooking and eating meat. Common limitations of these
products are a texture and mouthfeel that are more homogenous than
that of equivalent meat products. Furthermore, as the products must
largely be sold pre-cooked, with artificial flavors and aromas
built in, they fail to replicate aromas, flavors, and other key
features associated with cooking meat. As a result, these products
appeal mainly to a limited consumer base that is already committed
to vegetarianism/veganism, but have failed to appeal to the larger
consumer segment accustomed to eating meat.
[0006] Disclosed herein are improved methods and compositions which
more accurately replicate the characteristics that consumers value
in the preparation and consumption of meat and which overcome the
shortcomings and drawbacks of current meat substitutes.
SUMMARY OF THE INVENTION
[0007] In some aspects, the invention provides a meat substitute
composition comprising a protein content, wherein one or more
isolated and purified proteins accounts for 10% or more of said
protein content by weight, wherein said meat substitute composition
accurately mimics the taste, texture, or color of a meat product
derived from animal sources.
[0008] In one embodiment, the meat substitute composition
accurately mimics the color of said meat product in its raw state
and in a cooked state after cooking.
[0009] In another embodiment, the one or more isolated and purified
proteins accounts for 25% or more of said protein content by
weight.
[0010] In another embodiment, the one or more isolated and purified
proteins accounts for 50% or more of said protein content by
weight.
[0011] In another embodiment, the one or more isolated and purified
proteins accounts for 75% or more of said protein content by
weight.
[0012] In another embodiment, the one or more isolated and purified
proteins accounts for 90% or more of said protein content by
weight.
[0013] In another embodiment, gluten does not account for 10% or
more of said protein content by weight.
[0014] In another embodiment, each of said isolated, purified
proteins is separately isolated and purified.
[0015] In another embodiment, the meat substitute composition
comprises 1-7 isolated and purified proteins.
[0016] In another embodiment, said 1-7 isolated and purified
proteins are each isolated from different plant species.
[0017] In some embodiments the meat substitute comprises less than
5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20
individual proteins isolated from one or more plant species.
[0018] In another embodiment, said protein content comprises no
more than trace amounts of any other proteins derived from the one
or more plant species.
[0019] In another embodiment, said one or more isolated and
purified proteins are selected from the group consisting of
leghemoglobin, non-symbiotic hemoglobin, hemoglobin, myoglobin,
chlorocruorin, erythrocruorin, neuroglobin, cytoglobin,
protoglobin, truncated 2/2 globin, HbN, cyanoglobin, HbO, Glb3, and
cytochromes, Hell's gate globin I, bacterial hemoglobins, ciliate
myoglobins, flavohemoglobins, ribosomal proteins, actin,
hexokinase, lactate dehydrogenase, fructose bisphosphate aldolase,
phosphofructokinases, triose phosphate isomerases, phosphoglycerate
kinases, phosphoglycerate mutases, enolases, pyruvate kinases,
glyceraldehyde-3-phosphate dehydrogenases, pyruvate decarboxylases,
actins, translation elongation factors, ribulose-1,5-bisphosphate
carboxylase oxygenase (rubisco), ribulose-1,5-bisphosphate
carboxylase oxygenase activase (rubisco activase), albumins,
glycinins, conglycinins, globulins, vicilins, conalbumin, gliadin,
glutelin, gluten, glutenin, hordein, prolamin, phaseolin (protein),
proteinoplast, secalin, extensins, triticeae gluten, zein, any seed
storage protein, oleosins, caloleosins, steroleosins or other oil
body proteins, vegetative storage protein A, vegetative storage
protein B, moong seed storage 8S globulin.
[0020] In another embodiment, said one or more isolated and
purified proteins are not isolated from an animal.
[0021] In another embodiment, said one or more isolated and
purified proteins are isolated from a single plant source.
[0022] In another embodiment, said one or more isolated and
purified proteins are isolated from multiple plant sources.
[0023] In another embodiment, wherein said one or more isolated,
purified proteins are isolated from a genetically modified
organism.
[0024] In some embodiments, said genetically modified organism is a
genetically modified bacteria or yeast organism.
[0025] In some embodiments, said isolated, purified protein has
been formed into fibers.
[0026] In particular embodiments, said fibers resemble skeletal
muscle fibers.
[0027] In yet more particular embodiments, said fibers are
asymmetric fibers.
[0028] In some embodiments, the meat substitute composition further
comprises one or more isolated and purified iron-containing
proteins.
[0029] In some embodiments, said one or more isolated and purified
iron-containing proteins is selected from the group consisting of
hemoglobin, myoglobin, leghemoglobin, non-symbiotic hemoglobin,
chlorocruorin, erythrocruorin, neuroglobin, cytoglobin,
protoglobin, truncated 2/2 globin, HbN, cyanoglobin, HbO, Glb3, and
Hell's gate globin I, bacterial hemoglobins, ciliate myoglobins,
flavohemoglobins.
[0030] In a particular embodiment, said iron-containing protein
comprises an amino acid sequence with at least 70% homology to SEQ
ID NO 1. SEQ ID NO 1:
MVAFTEKQDALVSSSFEAFKANIPQYSVVFYTSILEKAPAAKDLFSFLANGVDPTNPKLTGHAE
KLFALVRDSAGQLKASGTVVADAALGSVHAQKAVTDPQFVVVKEALLKTIKAAVGDKWSDE
LSRAWEVAYDELAAAIKKA.
[0031] In a particular embodiment, said iron-containing protein
comprises an amino acid sequence with at least 70% homology to SEQ
ID NO 2. SEQ ID NO 2: MIDQKEKELI KESWKRIEPN KNEIGLLFYA NLFKEEPTVS
VLFQNPISSQ SRKLMQVLGI LVQGIDNLEG LIPTLQDLGR RHKQYGVVDS HYPLVGDCLL
KSIQEYLGQG FTEEAKAAWT KVYGIAAQVM TAE. In some embodiments said
iron-containing protein comprises an amino acid sequence with at
least 80% homology to SEQ ID NO 2. In some embodiments said
iron-containing protein comprises an amino acid sequence with at
least 90% homology to SEQ ID NO 2. In some embodiments said
iron-containing protein comprises an amino acid sequence with at
least 98% homology to SEQ ID NO 2.
[0032] In a particular embodiment, said iron-containing protein
comprises an amino acid sequence with at least 70% homology to SEQ
ID NO 3. SEQ ID NO 3: MRKQPTVFEK LGGQAAMHAA VPLFYKKVLA DDRVKHYFKN
TNMEHQAKQQ EDFLTMLLGG PNHYKGKNMA EAHKGMNLQN SHFDAIIENL AATLKELGVS
DQIIGEAAKV IEHTRKDCLG K. In some embodiments said iron-containing
protein comprises an amino acid sequence with at least 80% homology
to SEQ ID NO 3. In some embodiments said iron-containing protein
comprises an amino acid sequence with at least 90% homology to SEQ
ID NO 3. In some embodiments said iron-containing protein comprises
an amino acid sequence with at least 98% homology to SEQ ID NO
3.
[0033] In some embodiments, the isolated and purified proteins are
assembled into one or more gels.
[0034] In some embodiments, the meat substitute composition further
comprises one or more fats.
[0035] In particular embodiments, said one or more fats are derived
from a plant source.
[0036] In another aspect, the invention provides a meat substitute
product that comprises an indicator that indicates cooking
progression from a raw state to a cooked state, wherein said meat
substitute product is derived from non-animal sources.
[0037] In some embodiments, said indicator is a visual indicator
that accurately mimics the color transition of a meat product
during said cooking progression.
[0038] In one embodiment, said color transition is from red to
brown.
[0039] In one embodiment, said color transition is from pink to
white or tan.
[0040] In one embodiment, said visual indicator transitions from a
translucent to opaque color during said cooking progression.
[0041] In some embodiments, the indicator is an olfactory indicator
that indicates cooking progression.
[0042] In one embodiment, said olfactory indicator is one or more
volatile odorants released during cooking.
[0043] In some embodiments, said indicator comprises one or more
isolated, purified iron-containing proteins.
[0044] In particular embodiments, said one or more isolated,
purified iron-containing proteins is in a reduced state before
cooking.
[0045] In one embodiment, said one or more isolated and purified
iron-containing proteins is selected from the group consisting of
hemoglobin, myoglobin, leghemoglobin, non-symbiotic hemoglobin,
chlorocruorin, erythrocruorin, neuroglobin, cytoglobin,
protoglobin, truncated 2/2 globin, HbN, cyanoglobin, HbO, Glb3, and
cytochromes, Hell's gate globin I, bacterial hemoglobins, ciliate
myoglobins, flavohemoglobins.
[0046] In a particular embodiment, said iron-containing protein
comprises an amino acid sequence with at least 70% homology to SEQ
ID NO 1. SEQ ID NO 1:
MVAFTEKQDALVSSSFEAFKANIPQYSVVFYTSILEKAPAAKDLFSFLANGVDPTNPKLTGHAE
KLFALVRDSAGQLKASGTVVADAALGSVHAQKAVTDPQFVVVKEALLKTIKAAVGDKWSDE
LSRAWEVAYDELAAAIKKA.
[0047] In some embodiments, said one or more isolated and purified
iron-containing proteins are not isolated from an animal. In some
embodiments compositions of the invention do not contain any
proteins from an animal.
[0048] In particular embodiments, said one or more isolated and
purified iron-containing proteins are isolated from one or more
plant sources.
[0049] In yet more particular embodiments, said one or more
isolated, purified proteins are isolated from the root nodules,
roots, seeds, leaves, or stems of said one or more plant
sources.
[0050] In other particular embodiments, said one or more plant
sources are soy or pea plants.
[0051] In one embodiment, said one or more plant sources comprises
one or more plants of the legume family.
[0052] In some embodiments, said one or more isolated and purified
iron carrying proteins in a reduced or oxidized state has a similar
UV-VIS profile to a myoglobin protein derived from an animal source
when in an equivalent reduced or oxidized state.
[0053] In a particular embodiment, the difference between the peak
absorbance wavelength of said one or more isolated and purified
iron-containing proteins and the peak absorbance wavelength of
myoglobin derived from an animal source is less than 5%.
[0054] In some embodiments, said one or more isolated, purified
proteins are isolated from a genetically modified organism.
[0055] In one embodiment, said genetically modified organism is a
genetically modified bacteria or yeast organism.
[0056] In some embodiments, the meat substitute product contains no
methylcellulose, no carrageenan, no caramel color, no konjac flour,
no gum arabic, and no acacia gum.
[0057] In particular embodiments, the meat substitute product
additionally contains less than 1% wheat gluten.
[0058] In a more particular embodiment, said meat substitute
product contains no wheat gluten.
[0059] In other particular embodiments, said meat substitute
product contains no soy protein isolate.
[0060] In other particular embodiments, said meat substitute
product contains no soy protein concentrate.
[0061] In other particular embodiments, said meat substitute
product contains no soy protein.
[0062] In a more particular embodiment, said meat substitute
product contains less than 5% carbohydrates.
[0063] In other particular embodiments, said meat substitute
product contains no tofu.
[0064] In some embodiments, said meat substitute product contains
no tofu, and no wheat gluten.
[0065] In some embodiments, said meat substitute product contains
no soy protein, and no wheat gluten.
[0066] In some embodiments, said meat substitute product contains
no animal products and less than 5% carbohydrates.
[0067] In some embodiments, said meat substitute product contains
less than 1% cellulose.
[0068] In some embodiments, said meat substitute product contains
less than 5% insoluble carbohydrates.
[0069] In some embodiments, said meat substitute product contains
no soy protein, and less than 1% cellulose.
[0070] In some embodiments, said meat substitute product contains
no soy protein, and less than 5% insoluble carbohydrates.
[0071] In some embodiments, said meat substitute product contains
no wheat gluten, and less than 1% cellulose.
[0072] In some embodiments, said meat substitute product contains
no wheat gluten, and less than 5% insoluble carbohydrates.
[0073] In another aspect, the invention provides a muscle tissue
replica comprising a protein content, wherein said protein content
comprises one or more isolated and purified proteins, wherein said
muscle tissue replica approximates the taste, texture, or color of
an equivalent muscle tissue derived from an animal source.
[0074] In some embodiments, said one or more isolated and purified
proteins accounts for at least 50% of said protein content by
weight. In some embodiments, said one or more isolated and purified
proteins accounts for at least 40% of said protein content by
weight. In some embodiments, said one or more isolated and purified
proteins accounts for at least 30% of said protein content by
weight. In some embodiments, said one or more isolated and purified
proteins accounts for at least 20% of said protein content by
weight. In some embodiments, said one or more isolated and purified
proteins accounts for at least 10% of said protein content by
weight.
[0075] In some embodiments, said one or more isolated and purified
proteins accounts for at least 50% of said composition content by
weight. In some embodiments, said one or more isolated and purified
proteins accounts for at least 40% of said composition content by
weight. In some embodiments, said one or more isolated and purified
proteins accounts for at least 30% of said composition content by
weight. In some embodiments, said one or more isolated and purified
proteins accounts for at least 20% of said composition content by
weight. In some embodiments, said one or more isolated and purified
proteins accounts for at least 10% of said composition content by
weight. In some embodiments, said one or more isolated and purified
proteins accounts for at least 5% of said composition content by
weight. In some embodiments, said one or more isolated and purified
proteins accounts for at least 1% of said composition content by
weight.
[0076] In some embodiments, said protein content is derived from
one or more non-animal sources.
[0077] In particular embodiments, said one or more non-animal
sources is a plant source.
[0078] In other particular embodiments, said one or more non-animal
sources is a genetically modified yeast or bacteria.
[0079] In some embodiments, each of said one or more isolated
proteins is isolated and purified separately.
[0080] In some embodiments, said one or more isolated proteins are
selected from the group consisting of hemoglobin, myoglobin,
chlorocruorin, erythrocruorin, neuroglobin, cytoglobin,
protoglobin, truncated 2/2 globin, HbN, cyanoglobin, HbO, Glb3, and
cytochromes, Hell's gate globin I, bacterial hemoglobins, ciliate
myoglobins, flavohemoglobins, ribosomal proteins, actin,
hexokinase, lactate dehydrogenase, fructose bisphosphate aldolase,
phosphofructokinases, triose phosphate isomerases, phosphoglycerate
kinases, phosphoglycerate mutases, enolases, pyruvate kinases,
glyceraldehyde-3-phosphate dehydrogenases, pyruvate decarboxylases,
actins, translation elongation factors, ribulose-1,5-bisphosphate
carboxylase oxygenase (rubisco), ribulose-1,5-bisphosphate
carboxylase oxygenase activase (rubisco activase), albumins,
glycinins, conglycinins, globulins, vicilins, conalbumin, gliadin,
glutelin, gluten, glutenin, hordein, prolamin, phaseolin (protein),
proteinoplast, secalin, extensins, triticeae gluten, zein, any seed
storage protein, oleosins, caloleosins, steroleosins or other oil
body proteins, vegetative storage protein A, vegetative storage
protein B, moong seed storage 8S globulin.
[0081] In one embodiment, said seed storage protein is moong bean
8S protein.
[0082] In some embodiments, said protein content is suspended in a
gel.
[0083] In some embodiments, said protein content is in the form of
a gel.
[0084] In one embodiment, said gel comprises an isolated, purified
cross-linking enzyme.
[0085] In some embodiments, said isolated, purified cross-linking
enzyme is selected from the group consisting of transglutaminase,
lysyl oxidases, and amine oxidases.
[0086] In a particular embodiment, said isolated, purified
cross-linking enzyme is transglutaminase.
[0087] In some embodiments, said protein content has been assembled
into fibers.
[0088] In particular embodiments, said fibers are arranged
isotropically.
[0089] In one embodiment, said fibers are asymmetric fibers.
[0090] In some embodiments, the muscle tissue replica further
comprises one or more isolated and purified iron-containing
proteins.
[0091] In some embodiments, said one or more isolated and purified
iron-containing proteins is selected from the group consisting of
hemoglobin, myoglobin, leghemoglobin, non-symbiotic hemoglobin,
chlorocruorin, erythrocruorin, neuroglobin, cytoglobin,
protoglobin, truncated 2/2 globin, HbN, cyanoglobin, HbO, Glb3, and
cytochromes, Hell's gate globin I, bacterial hemoglobins, ciliate
myoglobins, flavohemoglobins.
[0092] In a particular embodiment, said one or more isolated and
purified iron-containing proteins comprises an amino acid sequence
with at least 70% homology to SEQ ID NO 1. SEQ ID NO 1:
MVAFTEKQDALVSSSFEAFKANIPQYSVVFYTSILEKAPAAKDLFSFLANGVDPTNPKLTGHAE
KLFALVRDSAGQLKASGTVVADAALGSVHAQKAVTDPQFVVVKEALLKTIKAAVGDKWSDE
LSRAWEVAYDELAAAIKKA. In a particular embodiment, said one or more
isolated and purified iron-containing proteins comprises an amino
acid sequence with at least 80% homology to SEQ ID NO 1. In a
particular embodiment, said one or more isolated and purified
iron-containing proteins comprises an amino acid sequence with at
least 90% homology to SEQ ID NO 1. In a particular embodiment, said
one or more isolated and purified iron-containing proteins
comprises an amino acid sequence with at least 95% homology to SEQ
ID NO 1. In a particular embodiment, said one or more isolated and
purified iron-containing proteins comprises an amino acid sequence
with at least 98% homology to SEQ ID NO 1.
[0093] In particular embodiments, the muscle tissue replica
comprises a protein content, wherein (i) one isolated and purified
protein that is not an iron-containing protein accounts for 40-95%
of said protein content, (ii) one or more isolated and purified
iron-containing proteins accounts for 1-20% of said protein
content, and (iii) one or more cross-linking reagents accounts for
0.1-35% of said protein content.
[0094] In one embodiment, said protein content accounts for 5-50%
of said replica by weight or by weight/volume.
[0095] In one embodiment, said one isolated and purified protein is
moong bean 8S protein.
[0096] In one embodiment, said one or more isolated and purified
iron-containing proteins is leghemoglobin.
[0097] In one embodiment, said one or more cross-linking reagents
is transglutaminase.
[0098] In some embodiments, the muscle tissue replica contains no
methylcellulose, no carrageenan, no caramel color, no konjac flour,
no gum arabic, and no acacia gum.
[0099] In particular embodiments, the muscle tissue replica
additionally contains less than 1% wheat gluten. In particular
embodiments, the muscle tissue replica additionally contains less
than 5% wheat gluten. In particular embodiments, the muscle tissue
replica additionally contains less than 10% wheat gluten. In
particular embodiments, the muscle tissue replica additionally
contains less than 0.1% wheat gluten.
[0100] In a more particular embodiment, said muscle tissue replica
contains no wheat gluten.
[0101] In other particular embodiments, said muscle tissue replica
contains no soy protein isolate.
[0102] In other particular embodiments, said muscle tissue replica
contains no soy protein concentrate.
[0103] In other particular embodiments, said muscle tissue replica
contains no soy protein.
[0104] In a more particular embodiment, said muscle tissue replica
contains less than 5% carbohydrates.
[0105] In other particular embodiments, said muscle tissue replica
contains no tofu.
[0106] In some embodiments, said muscle tissue replica contains no
tofu, and no wheat gluten.
[0107] In some embodiments, said muscle tissue replica contains no
soy protein, and no wheat gluten.
[0108] In some embodiments, said muscle tissue replica contains no
animal products and less than 5% carbohydrates.
[0109] In some embodiments, said muscle tissue replica contains
less than 1% cellulose.
[0110] In some embodiments, said muscle tissue replica contains
less than 5% insoluble carbohydrates.
[0111] In some embodiments, said muscle tissue replica contains no
soy protein, and less than 1% cellulose.
[0112] In some embodiments, said muscle tissue replica contains no
soy protein, and less than 5% insoluble carbohydrates.
[0113] In some embodiments, said muscle tissue replica contains no
wheat gluten, and less than 1% cellulose.
[0114] In some embodiments, said muscle tissue replica contains no
wheat gluten, and less than 5% insoluble carbohydrates.
[0115] In some embodiments, the muscle tissue replica contains no
methylcellulose, no carrageenan, no caramel color, no konjac flour,
no gum arabic, and no acacia gum.
[0116] In particular embodiments, the meat replica contains less
than 1% wheat gluten. In particular embodiments, the meat replica
contains less than 5% wheat gluten. In particular embodiments, the
meat replica contains less than 10% wheat gluten. In particular
embodiments, the meat replica contains less than 0.1% wheat
gluten.
[0117] In a more particular embodiment, the meat replica contains
no wheat gluten.
[0118] In other particular embodiments, the meat replica contains
no soy protein isolate.
[0119] In other particular embodiments, the meat replica contains
no soy protein concentrate.
[0120] In other particular embodiments, the meat replica contains
no soy protein.
[0121] In a more particular embodiment, the meat replica contains
less than 5% carbohydrates.
[0122] In other particular embodiments, the meat replica contains
no tofu.
[0123] In some embodiments, the meat replica contains no tofu, and
no wheat gluten.
[0124] In some embodiments, the meat replica contains no soy
protein, and no wheat gluten.
[0125] In some embodiments, the meat replica contains no animal
products and less than 5% carbohydrates.
[0126] In some embodiments, the meat replica contains less than 1%
cellulose. In some embodiments, the meat replica contains less than
0.1% cellulose. In some embodiments, the meat replica contains less
than 10% cellulose. In some embodiments, the meat replica contains
less than 5% cellulose.
[0127] In some embodiments, the meat replica contains less than 5%
insoluble carbohydrates.
[0128] In some embodiments, the meat replica contains no soy
protein, and less than 1% cellulose.
[0129] In some embodiments, the meat replica contains no soy
protein, and less than 5% insoluble carbohydrates.
[0130] In some embodiments, the meat replica contains no wheat
gluten, and less than 1% cellulose.
[0131] In some embodiments, the meat replica contains no wheat
gluten, and less than 5% insoluble carbohydrates.
[0132] In another aspect, the invention provides a fat tissue
replica comprising a gelled emulsion, said gelled emulsion
comprising a protein solution with fat droplets suspended
therein.
[0133] In some embodiments, said fat droplets are derived from a
non-animal source.
[0134] In some embodiments, said fat droplets are comprised of one
or more plant oils.
[0135] In some embodiments, said one or more plant oils is selected
from the group consisting of corn oil, olive oil, soy oil, peanut
oil, walnut oil, almond oil, sesame oil, cottonseed oil, rapeseed
oil, canola oil, safflower oil, sunflower oil, flax seed oil, algal
oil, palm oil, palm kernel oil, coconut oil, babassu oil, shea
butter, mango butter, cocoa butter, wheat germ oil, rice bran oil,
oils produced by bacteria, algae, archaea or fungi or genetically
engineered bacteria, algae, archaea or fungi, triglycerides,
monoglycerides, diglycerides, sphingosides, glycolipids, lecithin,
lysolecithin, phosphatidic acids, lysophosphatidic acids, oleic
acid, palmitoleic acid, palmitic acid, myristic acid, lauric acid,
myristoleic acid, caproic acid, capric acid, caprylic acid,
pelargonic acid, undecanoic acid, linoleic acid, 20:1 eicosanoic
acid, arachidonic acid, eicosapentanoic acid, docosohexanoic acid,
18:2 conjugated linoleic acid, conjugated oleic acid, or esters of:
oleic acid, palmitoleic acid, palmitic acid, myristic acid, lauric
acid, myristoleic acid, caproic acid, capric acid, caprylic acid,
pelargonic acid, undecanoic acid, linoleic acid, 20:1 eicosanoic
acid, arachidonic acid, eicosapentanoic acid, docosohexanoic acid,
18:2 conjugated linoleic acid, or conjugated oleic acid, or
glycerol esters of oleic acid, palmitoleic acid, palmitic acid,
myristic acid, lauric acid, myristoleic acid, caproic acid, capric
acid, caprylic acid, pelargonic acid, undecanoic acid, linoleic
acid, 20:1 eicosanoic acid, arachidonic acid, eicosapentanoic acid,
docosohexanoic acid, 18:2 conjugated linoleic acid, or conjugated
oleic acid, or triglyceride derivatives of oleic acid, palmitoleic
acid, palmitic acid, myristic acid, lauric acid, myristoleic acid,
caproic acid, capric acid, caprylic acid, pelargonic acid,
undecanoic acid, linoleic acid, 20:1 eicosanoic acid, arachidonic
acid, eicosapentanoic acid, docosohexanoic acid, 18:2 conjugated
linoleic acid, or conjugated oleic acid.
[0136] In one embodiment, said one or more plant oils is rice bran
oil or canola oil.
[0137] In some embodiments, said protein solution comprises one or
more isolated, purified proteins.
[0138] In some embodiments, said one or more isolated, purified
proteins accounts for 75% or more of the protein in said protein
solution.
[0139] In some embodiments, said one or more isolated, purified
proteins are derived from a non-animal source.
[0140] In some embodiments, said non-animal source is a plant
source.
[0141] In some embodiments, said non-animal source is a genetically
modified yeast or bacteria.
[0142] In some embodiments, each of said one or more isolated
proteins is isolated and purified separately.
[0143] In some embodiments, said one or more isolated proteins are
selected from the group consisting of hemoglobin, myoglobin,
chlorocruorin, erythrocruorin, neuroglobin, cytoglobin,
protoglobin, truncated 2/2 globin, HbN, cyanoglobin, HbO, Glb3, and
cytochromes, Hell's gate globin I, bacterial hemoglobins, ciliate
myoglobins, flavohemoglobins, ribosomal proteins, actin,
hexokinase, lactate dehydrogenase, fructose bisphosphate aldolase,
phosphofructokinases, triose phosphate isomerases, phosphoglycerate
kinases, phosphoglycerate mutases, enolases, pyruvate kinases,
glyceraldehyde-3-phosphate dehydrogenases, pyruvate decarboxylases,
actins, translation elongation factors, ribulose-1,5-bisphosphate
carboxylase oxygenase (rubisco), ribulose-1,5-bisphosphate
carboxylase oxygenase activase (rubisco activase), albumins,
glycinins, conglycinins, globulins, vicilins, conalbumin, gliadin,
glutelin, gluten, glutenin, hordein, prolamin, phaseolin (protein),
proteinoplast, secalin, extensins, triticeae gluten, zein, any seed
storage protein, oleosins, caloleosins, steroleosins or other oil
body proteins, vegetative storage protein A, vegetative storage
protein B, moong seed storage 8S globulin.
[0144] In some embodiments, said one or more isolated, purified
proteins is an albumin protein, a seed storage protein, or pea
globulin protein.
[0145] In particular embodiments, said albumin protein is isolated
pea albumin protein.
[0146] In some embodiments, said seed storage protein is moong bean
8S protein.
[0147] In some embodiments, said gelled emulsion comprises a
protein solution comprising 1-3 isolated and purified proteins,
wherein said solution accounts for 30-70% of the volume of said
emulsion; a plant oil, wherein said plant oil accounts for 30-70%
of the volume of said emulsion; and an isolated, purified
cross-linking enzyme, wherein said cross-linking enzyme accounts
for 0.5-5% of said emulsion by wt/volume; wherein said plant oil is
emulsified in said protein solution, wherein said emulsion is
formed into a gel by said cross-linking enzyme.
[0148] In other embodiments said gelled emulsion comprises a
protein solution comprising 1-3 isolated and purified proteins,
wherein said solution accounts for 1-30% of the volume of said
emulsion; a plant oil, wherein said plant oil accounts for 70-99%
of the volume of said emulsion; and an isolated, purified
cross-linking enzyme, wherein said cross-linking enzyme accounts
for 0.5-5% of said emulsion by wt/volume; wherein said plant oil is
emulsified in said protein solution, wherein said emulsion is
formed into a gel by said cross-linking enzyme.
[0149] In some embodiments, the fat replica further comprises a
cross-linking enzyme.
[0150] In some embodiments, said cross-linking enzyme is
transglutaminase.
[0151] In some embodiments, one of said 1-3 isolated and purified
proteins is moong bean 8S protein, pea albumin protein, or pea
globulin protein.
[0152] In particular embodiments, said plant oil is rice bran oil
or canola oil.
[0153] In some embodiments, the fat tissue replica contains no
methylcellulose, no carrageenan, no caramel color, no konjac flour,
no gum arabic, and no acacia gum.
[0154] In particular embodiments, the fat tissue replica
additionally contains less than 1% wheat gluten.
[0155] In a more particular embodiment, said fat tissue replica
contains no wheat gluten.
[0156] In other particular embodiments, said fat tissue replica
contains no soy protein isolate.
[0157] In other particular embodiments, said fat tissue replica
contains no soy protein concentrate.
[0158] In other particular embodiments, said fat tissue replica
contains no soy protein.
[0159] In a more particular embodiment, said fat tissue replica
contains less than 5% carbohydrates.
[0160] In other particular embodiments, said fat tissue replica
contains no tofu.
[0161] In some embodiments, said fat tissue replica contains no
tofu, and no wheat gluten.
[0162] In some embodiments, said fat tissue replica contains no soy
protein, and no wheat gluten.
[0163] In some embodiments, said fat tissue replica contains no
animal products and less than 5% carbohydrates.
[0164] In some embodiments, said fat tissue replica contains less
than 1% cellulose.
[0165] In some embodiments, said fat tissue replica contains less
than 5% insoluble carbohydrates.
[0166] In some embodiments, said fat tissue replica contains no soy
protein, and less than 1% cellulose.
[0167] In some embodiments, said fat tissue replica contains no soy
protein, and less than 5% insoluble carbohydrates.
[0168] In some embodiments, said fat tissue replica contains no
wheat gluten, and less than 1% cellulose.
[0169] In some embodiments, said fat tissue replica contains no
wheat gluten, and less than 5% insoluble carbohydrates.
[0170] In another aspect, the invention provides a connective
tissue replica, comprising a protein content comprising one or more
isolated, purified proteins, wherein said protein content has been
assembled into structures approximating the texture and visual
appearance of connective tissue or skin.
[0171] In some embodiments, said protein content is derived from
non-animal source.
[0172] In some embodiments, said non-animal source is a plant
source.
[0173] In some embodiments, said non-animal source is a genetically
modified yeast or bacteria.
[0174] In some embodiments, said one or more isolated proteins
account for 50% or more of said protein content by weight.
[0175] In some embodiments, said one isolated and purified protein
accounts for 90% or more of said protein content by weight.
[0176] In some embodiments, each of said one or more isolated
proteins is isolated and purified separately.
[0177] In some embodiments, said one or more isolated proteins are
selected from the group consisting of hemoglobin, myoglobin,
chlorocruorin, erythrocruorin, neuroglobin, cytoglobin,
protoglobin, truncated 2/2 globin, HbN, cyanoglobin, HbO, Glb3, and
cytochromes, Hell's gate globin I, bacterial hemoglobins, ciliate
myoglobins, flavohemoglobins, ribosomal proteins, actin,
hexokinase, lactate dehydrogenase, fructose bisphosphate aldolase,
phosphofructokinases, triose phosphate isomerases, phosphoglycerate
kinases, phosphoglycerate mutases, enolases, pyruvate kinases,
glyceraldehyde-3-phosphate dehydrogenases, pyruvate decarboxylases,
actins, translation elongation factors, ribulose-1,5-bisphosphate
carboxylase oxygenase (rubisco), ribulose-1,5-bisphosphate
carboxylase oxygenase activase (rubisco activase), albumins,
glycinins, conglycinins, globulins, vicilins, conalbumin, gliadin,
glutelin, gluten, glutenin, hordein, prolamin, phaseolin (protein),
proteinoplast, secalin, extensins, triticeae gluten, zein, any seed
storage protein, oleosins, caloleosins, steroleosins or other oil
body proteins, vegetative storage protein A, vegetative storage
protein B, moong seed storage 8S globulin.
[0178] In some embodiments, said one or more isolated and purified
proteins is a prolamin family protein.
[0179] In some embodiments, said one or more isolated and purified
proteins is zein.
[0180] In some embodiments, said protein content is suspended in a
gel.
[0181] In some embodiments, said gel comprises an isolated,
purified cross-linking enzyme.
[0182] In some embodiments, said isolated, purified cross-linking
enzyme is selected from the group consisting of transglutaminase,
lysyl oxidases, and amine oxidases.
[0183] In some embodiments, said isolated, purified cross-linking
enzyme is transglutaminase.
[0184] In some embodiments, said protein content is formed into a
fiber.
[0185] In some embodiments, said fiber is produced by an extrusion
process.
[0186] In some embodiments, said fiber is stabilized by protein
crosslinks.
[0187] In some embodiments, fiber contains an isolated, purified
cross-linking enzyme.
[0188] In some embodiments, said isolated, purified cross-linking
enzyme is selected from the group consisting of transglutaminase,
lysyl oxidases, and amine oxidases.
[0189] In some embodiments, said isolated, purified cross-linking
enzyme is transglutaminase.
[0190] In another aspect, the invention provides a meat substitute
product, comprising a muscle replica; a fat tissue replica; and a
connective tissue replica; wherein said muscle replica, fat tissue
replica, and/or connective tissue replica are assembled in a manner
that approximates the physical organization of meat.
[0191] In some embodiments, the meat substitute product comprises
two or more of said muscle replica, fat tissue replica, and
connective tissue replica.
[0192] In some embodiments of the meat substitute product, said
muscle replica accounts for 40-90% of said product by weight, said
fat tissue replica accounts for 1-60% of said product by weight,
and said connective tissue replica accounts for 1-30% of said
product by weight.
[0193] In some embodiments, the meat substitute product comprises
60-90% water; 5-30% protein content; and 1-20% of a fat or fat
replica; wherein said protein content comprises one or more
isolated, purified plant proteins.
[0194] In some embodiments, said protein content is derived from
non-animal source.
[0195] In some embodiments, non-animal source is a plant
source.
[0196] In some embodiments, said non-animal source is a genetically
modified yeast or bacteria.
[0197] In some embodiments, 50% or more of said protein content by
weight are one or more isolated purified proteins.
[0198] In some embodiments, each of said one or more isolated
proteins is isolated and purified separately from different plant
species.
[0199] In some embodiments, one or more of said isolated proteins
is selected from the group consisting of: hemoglobin, myoglobin,
chlorocruorin, erythrocruorin, neuroglobin, cytoglobin,
protoglobin, truncated 2/2 globin, HbN, cyanoglobin, HbO, Glb3, and
cytochromes, Hell's gate globin I, bacterial hemoglobins, ciliate
myoglobins, flavohemoglobins, ribosomal proteins, actin,
hexokinase, lactate dehydrogenase, fructose bisphosphate aldolase,
phosphofructokinases, triose phosphate isomerases, phosphoglycerate
kinases, phosphoglycerate mutases, enolases, pyruvate kinases,
glyceraldehyde-3-phosphate dehydrogenases, pyruvate decarboxylases,
actins, translation elongation factors, ribulose-1,5-bisphosphate
carboxylase oxygenase (rubisco), ribulose-1,5-bisphosphate
carboxylase oxygenase activase (rubisco activase), albumins,
glycinins, conglycinins, globulins, vicilins, conalbumin, gliadin,
glutelin, gluten, glutenin, hordein, prolamin, phaseolin (protein),
proteinoplast, secalin, extensins, triticeae gluten, zein, any seed
storage protein, oleosins, caloleosins, steroleosins or other oil
body proteins, vegetative storage protein A, vegetative storage
protein B, moong seed storage 8S globulin.
[0200] In some embodiments, the meat substitute product further
comprises one or more isolated and purified iron-containing
proteins.
[0201] In some embodiments, said one or more isolated and purified
iron-containing proteins is selected from the group consisting of
hemoglobin, myoglobin, leghemoglobin, non-symbiotic hemoglobin,
chlorocruorin, erythrocruorin, neuroglobin, cytoglobin,
protoglobin, truncated 2/2 globin, HbN, cyanoglobin, HbO, Glb3, and
Hell's gate globin I, bacterial hemoglobins, ciliate myoglobins,
flavohemoglobins. In some embodiments, said iron-containing protein
comprises an amino acid sequence with at least 70% homology to SEQ
ID NO 1. SEQ ID NO 1:
MVAFTEKQDALVSSSFEAFKANIPQYSVVFYTSILEKAPAAKDLFSFLANGVDPTNPKLTGHAE
KLFALVRDSAGQLKASGTVVADAALGSVHAQKAVTDPQFVVVKEALLKTIKAAVGDKWSDE
LSRAWEVAYDELAAAIKKA
[0202] In some embodiments, the meat substitute product contains no
methylcellulose, no carrageenan, no caramel color, no konjac flour,
no gum arabic, and no acacia gum.
[0203] In particular embodiments, the meat substitute product
additionally contains less than 1% wheat gluten.
[0204] In a more particular embodiment, said meat substitute
product contains no wheat gluten.
[0205] In other particular embodiments, said meat substitute
product contains no soy protein isolate.
[0206] In other particular embodiments, said meat substitute
product contains no soy protein concentrate.
[0207] In other particular embodiments, said meat substitute
product contains no soy protein.
[0208] In a more particular embodiment, said meat substitute
product contains less than 5% carbohydrates.
[0209] In other particular embodiments, said meat substitute
product contains no tofu.
[0210] In some embodiments, said meat substitute product contains
no tofu, and no wheat gluten.
[0211] In some embodiments, said meat substitute product contains
no soy protein, and no wheat gluten.
[0212] In some embodiments, said meat substitute product contains
no animal products and less than 5% carbohydrates.
[0213] In some embodiments, said meat substitute product contains
less than 1% cellulose.
[0214] In some embodiments, said meat substitute product contains
less than 5% insoluble carbohydrates.
[0215] In some embodiments, said meat substitute product contains
no soy protein, and less than 1% cellulose.
[0216] In some embodiments, said meat substitute product contains
no soy protein, and less than 5% insoluble carbohydrates.
[0217] In some embodiments, said meat substitute product contains
no wheat gluten, and less than 1% cellulose.
[0218] In some embodiments, said meat substitute product contains
no wheat gluten, and less than 5% insoluble carbohydrates.
[0219] In another aspect, the invention provides a food product
comprising one or more isolated, purified iron-containing proteins,
wherein said food product is configured for consumption by an
animal.
[0220] In some embodiments, said one or more isolated, purified
iron-containing proteins is derived from a non-animal source.
[0221] In some embodiments, said non-animal source is a plant
source.
[0222] In some embodiments, said plant source comprises one or more
plants of the legume family.
[0223] In some embodiments, said one or more plants of the legume
family is a soy or pea plant.
[0224] In some embodiments, said non-animal source is a genetically
modified yeast or bacteria.
[0225] In some embodiments, said iron-containing protein is
selected from the group consisting of hemoglobin, myoglobin,
leghemoglobin, non-symbiotic hemoglobin, chlorocruorin,
erythrocruorin, neuroglobin, cytoglobin, protoglobin, truncated 2/2
globin, HbN, cyanoglobin, HbO, Glb3, and cytochromes, Hell's gate
globin I, bacterial hemoglobins, ciliate myoglobins,
flavohemoglobins.
[0226] In one embodiment, said iron-containing protein comprises an
amino acid sequence with at least 70% homology to SEQ ID NO 1. SEQ
ID NO 1:
MVAFTEKQDALVSSSFEAFKANIPQYSVVFYTSILEKAPAAKDLFSFLANGVDPTNPKLTGHAE
KLFALVRDSAGQLKASGTVVADAALGSVHAQKAVTDPQFVVVKEALLKTIKAAVGDKWSDE
LSRAWEVAYDELAAAIKKA
[0227] In some embodiments, the food product contains no
methylcellulose, no carrageenan, no caramel color, no konjac flour,
no gum arabic, and no acacia gum.
[0228] In particular embodiments, the food product additionally
contains less than 1% wheat gluten.
[0229] In a more particular embodiment, said food product contains
no wheat gluten.
[0230] In other particular embodiments, said food product contains
no soy protein isolate.
[0231] In other particular embodiments, said food product contains
no soy protein concentrate.
[0232] In other particular embodiments, said food product contains
no soy protein.
[0233] In a more particular embodiment, said food product contains
less than 5% carbohydrates.
[0234] In other particular embodiments, said food product contains
no tofu.
[0235] In some embodiments, said food product contains no tofu, and
no wheat gluten.
[0236] In some embodiments, said food product contains no soy
protein, and no wheat gluten.
[0237] In some embodiments, said food product contains no animal
products and less than 5% carbohydrates.
[0238] In some embodiments, said food product contains less than 1%
cellulose.
[0239] In some embodiments, said food product contains less than 5%
insoluble carbohydrates.
[0240] In some embodiments, said food product contains no soy
protein, and less than 1% cellulose.
[0241] In some embodiments, said food product contains no soy
protein, and less than 5% insoluble carbohydrates.
[0242] In some embodiments, said food product contains no wheat
gluten, and less than 1% cellulose.
[0243] In some embodiments, said food product contains no wheat
gluten, and less than 5% insoluble carbohydrates.
[0244] In another aspect, the invention provides a method of making
a meat substitute composition, comprising isolating and purifying
one or more proteins; and assembling said one or more proteins into
a physical organization that approximates the physical organization
of meat.
[0245] In another aspect, the invention provides a method of making
a muscle tissue replica, comprising isolating and purifying one or
more proteins; and assembling said one or more proteins into a
physical organization that approximates the physical organization
of skeletal muscle.
[0246] In another aspect, the invention provides a method of making
a fat tissue replica, comprising isolating and purifying one or
more proteins; preparing a solution comprising one or more
proteins; emulsifying one or more fats in said solution; and
stabilizing said solution into a gelled emulsification with one or
more cross-linking reagents.
[0247] In another aspect, the invention provides a method of making
a connective tissue replica, comprising isolating and purifying one
or more proteins; and precipitating said one or more proteins,
wherein said precipitating results in said one or more proteins
forming physical structures approximating the physical organization
of connective tissue.
[0248] In some embodiments, said precipitating comprises
solubilizing said one or more proteins in a first solution; and
extruding said first solution into a second solution, wherein said
one or more proteins is insoluble in said second solution, wherein
said extruding induces precipitation of said one or more
proteins.
[0249] In another aspect, the invention provides a food product
comprising one or more isolated, purified iron-containing proteins,
wherein said food product is configured for consumption by an
animal.
[0250] In another aspect, the invention provides a food product
comprising one or more isolated, purified iron-containing proteins,
wherein said food product is configured for consumption by
humans.
[0251] In another aspect, the invention provides a food product
comprising one or more isolated, purified iron-containing proteins,
wherein said food product is configured for consumption by an
animal. In another aspect, the invention provides a food product
comprising one or more isolated, purified iron-containing proteins,
wherein said food product is configured for consumption by
humans.
[0252] All publications and patent applications mentioned in this
specification are herein incorporated by reference to the same
extent as if each individual publication or patent application was
specifically and individually indicated to be incorporated by
reference.
BRIEF DESCRIPTION OF THE DRAWINGS
[0253] The patent or application file contains at least one drawing
executed in color. Copies of this patent or patent application
publication with color drawing(s) will be provided by the Office
upon request and payment of the necessary fee.
[0254] The novel features of the invention are set forth with
particularity in the appended claims. A better understanding of the
features and advantages of the present invention will be obtained
by reference to the following detailed description that sets forth
illustrative embodiments, in which the principles of the invention
are utilized, and the accompanying drawings of which:
[0255] FIG. 1 depicts a portion of the root of a pea plant (Pisum
sativum) with the root nodules sliced open to demonstrate the red
color conferred by leghemoglobin contained therein. The sliced open
rood nodule appears red.
[0256] FIG. 2 depicts leghemoglobin isolated from 1 oz of pea
roots. The red color commonly attributed to meat is evident in the
color photo.
[0257] FIG. 3 shows that leghemoglobins from different species are
homologs and have similar color properties. In FIG. 3, panel A
shows an SDS_PAGE gels of lysed root-nodules of three legume plant
species (1) Fava bean (2) English Pea (3) Soybean. Arrows mark
respective leghemoglobins. Panel B shows the similarity of UV-VIS
spectral profile of leghemoglobins from two different plant species
(Favabean and Soybean).
[0258] FIG. 4 shows a comparison of reduced (heme iron 2+) and
oxidized (heme iron 3+) soybean leghemoglobin (FIG. 4 panel A) and
equine heart muscle myoglobin (FIG. 4 panel B) showing similarity
of UV-VIS absorption profiles of two proteins. We purified soybean
leghemoglobin from soybean root-nodules using here described
protocol. Purified equine myoglobin was purchased from
SigmaAldrich. Soybean leghemoglobin (FIG. 4 panel A) and equine
myoglobin (FIG. 4 panel B) were reduced with 1 mm sodium
hydrosulfite. Shown are UV-VIS absorption spectra of heme Fe3+
(blue line--the higher peak in FIGS. 4 and 5) and heme Fe2+ (red
line) of soybean leghemoglobin (FIG. 4 panel A) and equine
myoglobin (FIG. 4 panel B). Insets show a zoom-in of UV-VIS spectra
in 450 nm to 700 nm region. (FIG. 4 panel C) Images of 10 ul liquid
droplet of a 40 mg/ml solution of soybean leghemoglobin in the
heme-Fe3+ state (left droplet) showing characteristic rusty red
color and a 40 mg/ml solution of soybean leghemoglobin solution in
the heme-Fe2+ state (right droplet) showing characteristic red
color of and (right image) corresponding samples of equine
myoglobin.
[0259] FIG. 5 depicts examples of successful reduction of
leghemoglobin heme iron with sodium hydrosulfite and titanium
citrate. In FIG. 5 the UV-VIS spectrogram of purified soybean
leghemoglobin in which the heme iron is in the oxidized (+3) state
is represented by the blue curves in each panel (the blue curves
have the higher peaks on the main graphs). The red curves in each
panel represent the UV-VIS spectra of the same leghemoglobin
species after reduction to the (+2) state (red lines) by addition
of (Panel A) 1 mM sodium hydrosulfite or (Panel B) 0.24% (wt/v)
titanium citrate in 20 mM potassium phosphate pH 7.3, 100 mM sodium
chloride buffer. The Insets show a zoom-in of UV-VIS spectra in
450-700 nm region. For this example, leghemoglobin was purified
from soybean root nodules using 60/90% ammonium sulfate
fractionation and exchanged into 20 mM potassium phosphate pH 7.4,
100 mM sodium chloride buffer. Sodium hydrosulfite stock solution
was prepared by dissolving 100 mM sodium hydrosulfite in 1 mM
sodium hydroxide in water. Titanium citrate stock solution was
prepared from 20% (wt/v) Ti-chloride in hydrochloric acid by mixing
it with 0.2M sodium citrate (1:10 v/v). pH was adjusted using
sodium carbonate to pH 7.0.
[0260] FIG. 6 depicts an example of the leghemoglobin purification
flow from soybean root nodules. The figure shows SDS-PAGE
fractionation of different soybean leghemoglobin purification steps
(Lane 1) Soybean root-nodule lysate; (Lane 2) Soybean root-nodule
lysate purified by 60/90% (wt/v) ammonium sulfate fractionation.
Shown is the protein content of 90% ammonium sulfate fractionated
protein pellet resuspended in 20 mm potassium phosphate pH 7.4, 100
mM sodium chloride, 1 mM EDTA buffer; Proteins from 90% ammonium
sulfate pellet were further purified by anion-exchange
chromatography (FFQ GE Healthcare) in 20 mM potassium phosphate ph
7.4, 100 mM sodium chloride. Leghemoglobin collected in the
flowthrough is shown in Lane 3. Anion-exchange flowthrough was
fractionated using size-exclusion chromatography (Sephacryl S-100
GE Healthcare) and resulting leghemoglobin fraction is shown in
Lane 4. Leghemoglobin content at different purification steps was
determined by determining the fraction of leghemoglobin band on
SDS-PAGE gel in a respective sample using ImageDoc analysis
software (BioRad). Purity (partial abundance) of leghemoglobin at
respective steps in the purification steps was: lysate: 32.7% (lane
1), 60/90% (wt/v) ammonium sulfate fractionation 78% (lane 2),
anion-exchange chromatography .about.83% (lane 3), and
size-exclusion chromatography to .about.95% (lane 4).
[0261] FIG. 7 shows stained SDS-PAGE gel analysis of (A) soybean
leghemoglobin expressed and purified using recombinant protein
technology and (B) soybean leghemoglobin purified from soybean root
nodules. (A) Recombinant Soybean leghemoglobin A carrying His-tag
and TEV protease His-tag removal site was expressed in E. coli BL21
strain and purified using His-tag affinity chromatography (Talon
resin, CloneTech). The left lane contains molecular weight
standards, the right lane contains purified recombinant soybean
leghemoglobin A (arrow). Expected molecular weight of the
recombinant soybean leghemoglobin A is 17.1 kDa. (B) SDS-PAGE gel
of purified Soybean leghemoglobin from root nodules. The left lane
contains molecular weight standards, the right lane contains
purified soybean leghemoglobin A (arrow). Mass spectrometry
analysis of purified material determined that all four soybean
leghemoglobin isoforms are present, and are full length (data not
shown). Expected molecular weights (MW) of soybean leghemoglobin
isoforms range from MW15.4 to 15.8 kDa.
[0262] FIG. 8 shows an example of 6 cubes of a commercial meat
analog (Quorn chicken analog), about 1 cm on a side, 4 of which
(Left and lower right) have been soaked in a solution of about 10
mg/ml soybean leghemoglobin in 20 mM Potassium phosphate pH 7.4 and
100 mM NaCl; the remaining two (Upper right) were soaked in the
same buffer without leghemoglobin. A deep pink color of the
leghemoglobin-infused cubes is apparent in color photos contrasting
the pale tan color of the un-infused cubes.
[0263] FIG. 9 shows the 4 leghemoglobin-infused cubes of Quorn
chicken analog in the process of cooking in a pan at 350.degree. C.
The two lower cubes have been turned over to expose the grilled
surface, which has turned brown. In the upper two cubes that the
heated portion has turned grey-brown, while the cooler top surface
retains its pink color. In some embodiments the consumable is
injected with a heme containing solution, for instance a
leghemoglobin solution, until the consumable is the color of
uncooked meat.
[0264] FIG. 10 depicts 43 ml of moong bean protein solution (150
mg/ml in dialysis buffer) were mixed with 37 ml of leghemoglobin
solution (46.5 mg/ml leghemoglobin and 20 mg/ml of other soybean
root nodule protein) in 20 mM potassium phosphate, 100 mM NaCl, pH
7.3). 20 ml of transglutaminase solution (20% w/w) were added,
solutions thoroughly mixed, divided into two 50 ml Falcon tubes and
incubated overnight at room temperature. Final protein
concentrations were 65 mg/ml for moong bean protein, 18 mg/ml of
leghemoglobin, 91 mg/ml total protein.
[0265] FIG. 11 depicts "White" muscle analog prepared by mixing 43
ml moong bean protein solution (150 mg/ml) with 45 ml of 11.7 mg/ml
solution of leghemoglobin and 0.8% (wt/v) of transglutaminase
solution. Final protein concentrations were 63 mg/ml for moong bean
protein, 5.2 mg/ml of leghemoglobin, 68 mg/ml total protein.
[0266] FIG. 12 depicts a fat tissue analog based on moong beans and
prepared in eppendorf tubes formed an opaque gel of off-white
color, smooth uniform texture, with no visible discernible liquid
that was not incorporated into the gel. The gel was freely
standing, elastic and springy. The gel has a slight, pleasant aroma
and a mild and pleasant flavor. The taste is mildly salty.
[0267] FIG. 13 depicts at tissue analog based on pea globulin and
prepared in eppendorf tubes very similar to moong bean-based fat
analog, except that it gave up a little of oil upon
compression.
[0268] FIG. 14 shows connective-tissue analog strands that were
created using a 1:3 ratio in 70% ethanol, loaded into a syringe
with a 23 gauge needle (ID 0.337 mm). The solution was slowly
extruded from the bottom of a 5 inch-high vessel into an excess of
5 M NaCl solution. The ethanol-zein solution being less dense than
the NaCl solution, floated upward, drawing out a fibrous stand of
solidifying zein. The NaCl was constantly stirred as the strands
began to develop to assist in the strand lengthening. The strands
bunch together and become a hard, dense mass.
[0269] FIG. 15 depicts a ground beef prototype patty was made by
combining 62% (wt/wt) muscle analog (62% (wt/wt) "dark muscle
analog" and 38% (wt/wt) "white muscle analog"), 29% (wt/wt) fat
tissue analog (from pea globulin and canola oil), 5% (wt/wt)
connective tissue analog (FIG. 15 panel A). A ground beef prototype
patty was made by combining 62% muscle analog (62% "dark muscle
analog" and 38% "white muscle analog), 29% fat tissue analog (from
moong bean seed 8S protein and rice bran oil), 5% connective tissue
analog (FIG. 15 panel B). A ground beef prototype patty was made by
combining 71% (wt/wt) muscle tissue analog (composed of 60% "white"
muscle analog, 40% "dark" muscle analog), 23% fat tissue (from pea
seed globulin proteins and canola oil) (FIG. 15 panel C). A ground
beef prototype patty was made by combining 67% "White" muscle
analog, with 28% fat tissue analog (from pea globulins and rice
bran oil), (FIG. 15, panel D)
[0270] FIG. 16 depicts a ground beef patty analog was made by
combining 62% (wt/wt) muscle tissue analog (62% (wt/wt) "dark
muscle analog" and 38% (wt/wt) "muscle analog"), 29% (wt/wt) fat
tissue analog (from pea globulin and canola oil), 5% (wt/wt)
connective tissue analog. The panel on the left shows the patty
before cooking and the panel on the right shows the same patty
after cooking for about 2 minutes. Observers described the aroma of
the cooking ground beef replica as distinctly "beefy".
DETAILED DESCRIPTION OF THE INVENTION
[0271] Methods and compositions for the production of consumables
are described herein. The consumables can be for animal
consumption. For example the consumable can be food fit for human
consumption. The consumable can be approved by suitable regulatory
authorities. The consumables can be sold in grocery stores or
prepared in restaurants, schools, hospitals, military facilities,
prisons, shelters, long-term care facilities, similar to already
existing human foods. The consumables could also be food for
domestic animals. For instance, dog food could be produced
according to the present inventions. The consumables may also be
food for wild animals. For instance, the consumables could be
provided to non-domesticated predatory animals.
[0272] The consumables of the present invention can compete with,
supplement or replace animal based foods. For instance the
consumables can be meat replicas made entirely from plant sources.
The consumables can be made to mimic the cut or appearance of meat
as it is currently sold. For instance a consumable may be visually
similar to or indistinguishable from ground beef or a particular
cut of beef. Alternatively, the consumables can be made with a
unique look or appearance. For instance the consumable could
contain patterns or lettering that is based upon the structure of
the consumable. In some instances the consumables look like
traditional meat products after they are prepared. For example a
consumable may be produced which is larger than a traditional cut
of beef but which, after the consumable is sliced and cooked
appears the same as a traditional cooked meet. In some embodiments
the consumable may resemble a traditional meat shape in two
dimensions, but not in a third. For example the consumable may
resemble a cut of meat in two dimensions (for example when viewed
from the top), but may be much longer (or thinker) than the
traditional cut. So in some embodiments a composition that can be
cut repeatedly into traditionally meat shaped products is
provided.
[0273] The consumable may be made entirely from plant based
sources. In some instances the consumable can be made from organic
sources. The consumables may also be made from a combination of
plant based sources and animal based sources. For instance, the
consumable may be a ground beef product supplemented with plant
based products of the invention.
[0274] The consumables can be made from local products. For
instance the consumables can be made from plants grown within a
certain radius of the eventual consumer. That radius could be 1,
10, 100, or 1000 miles for example. So, in some embodiments, the
invention provides a method for producing a meat replica which does
not contain products which have been shipped over 1, 10, 100, or
1000 miles prior to producing the meat replica.
[0275] The present invention provides methods for producing
consistent properties from the consumables when they are produced
from various sources. So, for example, a plant based meat replica
produced from local plants in Iowa, USA, will have substantially
similar taste, odor, and texture as a plant based meat replica
produced from local plants in Lorraine, France. This consistency
allows for methods for advertising locally grown foods with
consistent properties. The consistency can arise from the
concentration or purification of similar components at different
locations. These components can be combined in predetermined ratios
to insure consistency. In some embodiments a high degree of
characteristic consistency is possible using components (e.g.
isolated or concentrated proteins and fats) which come from the
same plant species. In some embodiments a high degree of
characteristic consistency is possible using components (e.g.
isolated or concentrated proteins and fats) which come from the
different plant species. In some embodiments the same proteins can
be isolated from different plant species. In some embodiments the
invention provides for a method comprising isolating similar plant
constituents from plant sources in different locations, assembling
in both locations compositions provided herein, and selling the
compositions, wherein the compositions assembled and sold at
different the geographic locations have consistent physical and
chemical properties. In some embodiments the isolated constituents
are from different plant populations in different locations. In
some embodiments one or more of the isolated constituents are
shipped to the separate geographic locations.
[0276] The consumables may require fewer resources to produce than
consumables produced from domesticated animals. Accordingly, the
present invention provides for meat replicates which require less
water or energy to produce than meat. For example a consumable can
require less than about 10, 50, 100, 200, 300, 500, or 1000 gallons
of water per pound of consumable. For comparison beef can require
over 2000 gallons of water per pound of meat.
[0277] The consumable may require less land are to produce than a
meat product with similar protein content. For example the
consumable may require 30% or less of the land area required to
produce a meat product with similar protein content.
[0278] The consumable may have health benefits compared to an
animal product it replaces in the diet. For example it may have
less cholesterol or lower levels of saturated fats than comparable
meat products.
[0279] The consumable may have animal welfare benefits compared to
an animal product it replaces in the diet. For instance it may be
produced without requiring confinement, forced feeding, premature
weaning, disruption of maternal-offspring interactions, or
slaughter of animals for their meat.
[0280] The consumable may have a smaller "carbon footprint" than
the meat products they replace. For example the consumable may
result in net greenhouse gas emissions of 1%, 5%, 10%, 25%, 50% or
75% of the greenhouse gas emissions attributable to the animal
product it replaces.
[0281] The consumable may provide alternatives to animal products
or combinations of animal products whose consumption is forbidden
by religious beliefs. For example, the consumable may be a kosher
pork chop.
[0282] The consumable can also be shipped in components and
produced or assembled at a different location. When available local
components can be used for production of the consumable. These can
be supplemented with components which are not locally available.
This allows for methods of producing consumables, for instance meat
replicates, using less energy in shipment than is required for
meat. For example, local water can be used in combination with a
kit which provides other components of the consumable. Using local
water will reduce shipping weight thereby reducing cost and
environmental impact.
[0283] The consumables can be produced or assembled wholly or in
part in areas where animal farming is not practical or is not
allowed. The consumable can be produced or assembled within an
urban environment. For example a kit may be provided to a user to
enable the user to produce the consumable. The user could use local
water or use plants from a rooftop garden, for instance in
Shanghai. In another example, the consumables could be produced
aboard a space craft, space station, or lunar base. Accordingly,
the present invention provides methods and systems for the
production of meat replicas for use in space travel or for training
for the same. For instance the present invention could be used in
earth based training for space travel. The consumables could also
be produced on an island or upon a manmade platform at sea where
the keeping of livestock is difficult or prohibited.
[0284] The consumables are, in some embodiments, designed to
replicate the experience of eating meat. The look, texture, and
taste of the consumable can be such that it is similar or
indistinguishable from meat. The invention therefore provides in
certain embodiments methods for determining whether an animal or
human can distinguish the consumable from meat.
[0285] One method to determine whether the consumable is comparable
to meat is to a) define the properties of meat and b) determine
whether the consumable has similar properties. Properties of meat
that can be tested include mechanical properties such as hardness,
cohesiveness, brittleness, chewiness, gumminess, viscosity,
elasticity, and adhesiveness. Properties of meat that can be tested
also include geometric properties such as particle size and shape,
and particle shape and orientation. Additional properties can
include moisture content and fat content. These properties can be
described using terms such as "soft," "firm" or "hard" describe
hardness; "crumbly," "crunchy," "brittle," "chewy," "tender,"
"tough," "short," "mealy," "pasty," or "gummy," to describe
cohesiveness; "thin" or "viscous" to describe viscosity; "plastic"
or "elastic" to describe elasticity; "sticky," "tacky" or "gooey"
to describe adhesiveness; "gritty," "grainy" or "course" to
describe particle shape and size; "fibrous," "cellular" or
"crystalline" to describe particle shape and orientation, "dry,"
"moist," "wet," or "watery" to describe moisture content; or "oily"
or "greasy" to describe fat content. So, in one embodiment a group
of people can be asked to rate a certain meat, for instance ground
beef, according to properties which describe the meat. These
ratings can be used as an indication of the properties of the meat.
The consumables of the present invention can then be compared to
the meat to determine how similar the consumable is to the meat. In
some instances the properties of the consumables are then altered
to make the consumable more similar to the meat. So, in some
embodiments, the consumable is rated similar to meat according to
human evaluation. In some embodiments the consumable is
indistinguishable from real meat to a human.
[0286] In some embodiments, subjects asked to identify the
consumable identify it as a form of meat. In some embodiments one
property of the compositions of the invention is that an animal,
for example a human, will identify the composition as a meat. In
some embodiments the human identifies the composition of the
invention as having properties equivalent to meat. In some
embodiments one or more properties of meat are equivalent according
to a human's perception. Such properties include the properties
that can be tested. In some embodiments a human identifies a
consumable of the present invention as more meat like than meat
substitutes found in the art.
[0287] In embodiments an experiment can demonstrate that consumable
s acceptable to consumers. A panel can be used to screen a variety
of consumables described herein. A number of human panelists can
tested multiple consumable samples, namely, natural meats vs. the
consumable compositions described herein. Variables such as fat
content can be standardized for example to 20% fat using lean and
fat meat mixes. Fat content can be determined using the Babcock for
meat method (S. S. Nielson, Introduction to the Chemical Analysis
of Foods (Jones & Bartlett Publishers, Boston, 1994)). Mixtures
of ground beef and consumables of the invention prepared according
to the procedure described herein can be formulated.
[0288] Panelists can be served samples in booths, under red lights
or under white light, in an open consumer panel. Samples can be
assigned random three-digit numbers and rotated in ballot position
to prevent bias. Panelists can be asked to evaluate samples for
tenderness, juiciness, texture, flavor, and overall acceptability
using a hedonic scale from 1=dislike extremely, to 9=like
extremely, with a median of 5=neither like nor, dislike. Panelists
can be encouraged to rinse their mouths with water between samples,
and given opportunity to comment on each sample.
[0289] The results of this experiment can indicate significant
differences (p<0.05) or similarities between the traditional
meats and the compositions of the invention.
[0290] These results will demonstrate that the compositions of the
invention are judged as acceptably equivalent to real meat
products. Additionally these results can demonstrate that
compositions of the invention are preferred by panelist over other
commercially available meat substitutes. So, in some embodiments
the present invention provides for consumables that are
significantly similar to traditional meats.
[0291] Consumables of the invention can also have similar physical
characteristics as traditional meat. In one embodiment the force
required to pierce a 1 inch thick structure (e.g. a patty) made of
a consumable of the invention with a fixed diameter steel rod is
not significantly different than the force required to pierce a 1
inch thick similar meat structure (e.g. a ground beef patty) with a
similar fixed diameter steel rod. Accordingly, the invention
provides for consumables with similar physical strength
characteristics to meat.
[0292] In some embodiments composition of the invention have a
similar cook loss characteristic as meat. In one embodiment a
consumable of the invention with a similar fat and protein content
as ground beef has the same reduction in size when cooked as real
ground beef. Similar similarities in size loss profiles can be
achieved for various compositions of consumables described herein
matched to various meats.
[0293] In some embodiments the consumable is compared to real meat
based upon olfactometer readings. In various embodiments the
olfactometer can be used to assess odor concentration and odor
thresholds, odor suprathresholds with comparison to a reference
gas, hedonic scale scores to determine the degree of appreciation,
or relative intensity of odors. In some embodiments the
olfactometer allows the training and automatic evaluation of expert
panels. So in some embodiments the consumable is a product that
causes similar or identical olfactometer readings. In some
embodiments the similarity is sufficient to be beyond the detection
threshold of human perception.
[0294] Gas chromatography-mass spectrometry (GCMS) is a method that
combines the features of gas-liquid chromatography and mass
spectrometry to separate and identify different substances within a
test sample. GCMS can, in some embodiments, be used to evaluate the
properties of a consumable. For example volatile chemicals can be
isolated from the head space around meat. These chemicals can be
identified using GCMS. A profile of the volatile chemicals in the
headspace around meat is thereby created. In some instances each
peak of the GCMS can be further evaluated. For instance, a human
could rate the experience of smelling the chemical responsible for
a certain peak. This information could be used to further refine
the profile. GCMS could then be used to evaluate the properties of
the consumable. The GCMS profile could be used to refine the
consumable.
[0295] Characteristic flavor and fragrance components are mostly
produced during the cooking process by chemical reactions molecules
including amino acids, fats and sugars which are found in plants as
well as meat. Therefore in some embodiments the consumable is
tested for similarity to meat during or after cooking. In some
embodiments human ratings, human evaluation, olfactometer readings,
or GCMS measurements, or combinations thereof, are used to create
an olfactory map of cooked meat. Similarly, an olfactory map of the
consumable, for instance a meat replica, can be created. These maps
can be compared to assess how similar the cooked consumable it so
meat. In some embodiments the olfactory map of the consumable
during or after cooking is similar to or indistinguishable from
that of cooked or cooking meat. In some embodiments the similarity
is sufficient to be beyond the detection threshold of human
perception.
[0296] In one aspect, the invention provides a meat substitute
product (alternatively referred to herein as "consumable") that is
substantially or entirely composed of ingredients derived from
non-animal sources, yet recapitulates key features associated with
the cooking and consumption of an equivalent meat product derived
from animals. The equivalent meat product can be a white meat or a
dark meat. The equivalent meat product can be derived from any
animal. Non-limiting examples of animals used to derive the
equivalent meat product include farmed animals such as, e.g.,
cattle, sheep, pig, chicken, turkey, goose, duck, horse, dog or
game animals (whether wild or farmed) such as, e.g., rabbit, deer,
bison, buffalo, boar, snake, pheasant, quail, bear, elk, antelope,
pigeon, dove, grouse, fox, wild pig, goat, kangaroo, emu,
alligator, crocodile, turtle, groundhog, marmot, possum, partridge,
squirrel, raccoon, whale, seal, ostrich, capybara, nutria, guinea
pig, rat, mice, vole, any variety of insect or other arthropod,
seafood such as, e.g, fish, crab, lobster, oyster, muscle, scallop,
abalone, squid, octopus, sea urchin, tunicate and others. Many meat
products are typically derived from skeletal muscle of an animal
but it is understood that meat can also come from other muscles or
organs of the animal. In some embodiments, the equivalent meat
product is a cut of meat derived from skeletal muscle. In other
embodiments, the equivalent meat product is an organ such as, e.g.,
a kidney, heart, liver, gallbladder, intestine, stomach, bone
marrow, brain, thymus, lung, tongue. Accordingly, in some
embodiments the compositions of the present invention are
consumables similar to skeletal muscle or organs.
[0297] In some aspects, the present invention provides meat
substitute products comprising one or more of a first composition
comprising a muscle tissue replica, a second composition comprising
an adipose tissue replica, and/or a third composition comprising a
connective tissue replica, wherein the one or more compositions are
combined in a manner that recapitulates the physical organization
of meat. In other aspects, the present invention provides
compositions for a muscle tissue replica (herein referred to as
"muscle replica"), an adipose tissue replica (herein referred to as
"fat replica"), and a connective tissue replica (herein referred to
as "connective tissue replica"). In some embodiments, the
compositions and meat substitute products are principally or
entirely composed of ingredients derived from non-animal sources.
In alternative embodiments, the muscle, fat, and/or connective
tissue replica, or the meat substitute products comprising one or
more of said replicas, are partially derived from animal sources
but supplemented with ingredients derived from non-animal sources.
In yet other alternative embodiments, the invention provides meat
products substantially derived from animal sources but which are
supplemented with one or more of a muscle tissue replica, a fat
replica, and/or a connective tissue replica, wherein said replicas
are derived substantially or entirely from non-animal sources. A
non-limiting example of such a meat product is an ultra-lean ground
beef product supplemented with a non-animal derived fat replica
which improves texture and mouthfeel while preserving the health
benefits of a consumable low in animal fat. Such alternative
embodiments result in products with properties that more closely
recapitulate key features associated with preparing and consuming
meat but which are less costly and associated with a lesser
environmental impact, less animal welfare impact, or improved
health benefits for the consumer.
[0298] The physical organization of the meat substitute product can
be manipulated by controlling the localization, organization,
assembly, or orientation of the muscle, fat, and/or connective
tissue replicas described herein. In some embodiments the product
is designed in such a way that the replicas described herein are
associated with one another as in meat. In some embodiments the
consumable is designed so that after cooking the replicas described
herein are associated with one another as in cooked meat. In some
embodiments, one or more of the muscle, fat, and/or connective
tissue replicas are combined in a manner that recapitulate the
physical organization of different cuts or preparations of meat. In
an example embodiment, the replicas are combined in a manner that
approximates the physical organization of natural ground meat. In
other embodiments, the replicas are combined in a manner that
approximates different cuts of beef, such as, e.g., ribeye, filet
mignon, London broil, among others.
Proteins and Protein Sources
[0299] In some embodiments, any of the meat substitute products,
muscle tissue replica, fat replica, or connective tissue replica,
comprise one or more isolated, purified proteins. In some
embodiments, the meat substitute products are comprised of one or
more of a muscle replica, a fat replica, and/or connective tissue
replica which comprise one or more isolated, purified proteins. In
other embodiments, the muscle replica, fat replica, and/or
connective tissue replica comprises one or more isolated, purified
proteins. In some embodiments, about 0.1%, 0.2%, 0.5%, 1%, 2%, 3%,
4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%,
50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or more of
the protein component is comprised of one or more isolated,
purified proteins. For the purposes of this document, "purified
protein" will refer to a preparation in which the cumulative
abundance by mass of protein components other than the specified
protein, which can be a single monomeric or multimeric protein
species, is reduced by a factor of 2 or more, 3 or more, 5 or more,
10 or more, 20 or more, 50 or more, 100 or more or 1000 or more
relative to the source material from which the specified protein
was isolated.
[0300] In some embodiments, the one or more isolated, purified
proteins are derived from non-animal sources. Non-limiting examples
of non-animal sources include plants, funghi, bacteria, archaea,
genetically modified organisms such as genetically modified
bacteria or yeast, chemical or in vitro synthesis. In particular
embodiments, the one or more isolated, purified proteins are
derived from plant sources. Non-limiting examples of plant sources
include grains such as, e.g., corn, maize, rice, wheat, barley,
rye, triticale, teff, oilseeds including cottonseed, sunflower
seed, safflower seed, rapeseed, leafy greens such as, e.g.,
lettuce, spinach, kale, collard greens, turnip greens, chard,
mustard greens, dandelion greens, broccoli, cabbage, green matter
not ordinarily consumed by humans, including biomass crops,
including switchgrass, miscanthus, sorghum, other grasses, alfalfa,
corn stover, green matter ordinarily discarded from harvested
plants, sugar cane leaves, leaves of trees, root crops such as
cassava, sweet potato, potato, carrots, beets, turnips, plants from
the legume family, such as, e.g., clover, peas such as cowpeas,
english peas, yellow peas, green peas, beans such as, e.g.,
soybeans, fava beans, lima beans, kidney beans, garbanzo beans,
mung beans, pinto beans, lentils, lupins, mesquite, carob, soy, and
peanuts, vetch (vicia), stylo (stylosanthes), arachis, indigofera,
acacia, leucaena, cyamopsis, and sesbania. One of skill in the art
will understand that proteins that can be isolated from any
organism in the plant kingdom may be used in the present
invention.
[0301] Proteins that are abundant in plants can be isolated in
large quantities from one or more source plants and thus are an
economical choice for use in any of the muscle, fat, connective
tissue replicas, or meat substitute products. Accordingly, in some
embodiments, the one or more isolated proteins comprises an
abundant protein found in high levels in a plant and capable of
being isolated and purified in large quantities. In some
embodiments, the abundant protein comprises about 0.5%, 1%, 2%, 3%,
4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%,
50%, 55%, 60%, 65%, 70% of the total protein content of the source
plant. In some embodiments, the abundant protein comprises about
0.5-10%, about 5-40%, about 10-50%, about 20-60%, or about 30-70%
of the total protein content of the source plant. In some
embodiments, the abundant protein comprises about 0.5%, 1%, 2%, 3%,
4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%
of the total weight of the dry matter of the source plant. In some
embodiments, the abundant protein comprises about 0.5-5%, about
1-10%, about 5-20%, about 10-30%, about 15-40%, about 20-50% of the
total weight of the dry matter of the source plant.
[0302] In particular embodiments, the one or more isolated proteins
comprises an abundant protein that is found in high levels in the
leaves of plants. In some embodiments, the abundant protein
comprises about 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%,
20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80% of
the total protein content of the leaves of the source plant. In
some embodiments, the abundant protein comprises about 0.5-10%,
about 5%-40%, about 10%-60%, about 20%-60%, or about 30-70% of the
total protein content of the leaves of the source plant. In
particular embodiments, the one or more isolated proteins comprise
ribulose-1,5-bisphosphate carboxylase oxygenase (rubisco activase).
Rubisco is a particularly useful protein for meat replicas because
of its high solubility and an amino acid composition with close to
the optimum proportions of essential amino acids for human
nutrition. In particular embodiments, the one or more isolated
proteins comprise ribulose-1,5-bisphosphate carboxylase oxygenase
activase (rubisco activase). In particular embodiments, the one or
more isolated proteins comprise a vegetative storage protein
(VSP).
[0303] In some embodiments, the one or more isolated proteins
includes an abundant protein that is found in high levels in the
seeds of plants. In some embodiments, the abundant protein
comprises about 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%,
20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,
85% or 90% or more of the total protein content of the seeds of the
source plant. In some embodiments, the abundant protein comprises
about 0.5-10%, about 5%-40%, about 10%-60%, about 20%-60%, or about
30-70% or >70% of the total protein content of the seeds of the
source plant. Non-limiting examples of proteins found in high
levels in the seeds of plants are seed storage proteins, e.g.,
albumins, glycinins, conglycinins, globulins, vicilins, conalbumin,
gliadin, glutelin, gluten, glutenin, hordein, prolamin, phaseolin
(protein), proteinoplast, secalin, triticeae gluten, zein, any seed
storage protein, oleosins, caloleosins, steroleosins or other oil
body proteins
[0304] In some embodiments, the one or more isolated proteins
includes proteins that interact with lipids and help stabilize
lipids in a structure. Without wishing to be bound by a particular
theory, such proteins may improve the integration of lipids and/or
fat replicas with other components of the meat substitute product,
resulting in improved mouthfeel and texture of the final product. A
non-limiting example of a lipid-interacting plant protein is the
oleosin family of proteins. Oleosins are lipid-interacting proteins
that are found in oil bodies of plants. Other non-limiting examples
of plant proteins that can stabilize emulsions include seed storage
proteins from Great Northern Beans, albumins from peas, globulins
from peas, 8S globulins from Moong bean, 8S globulins from Kidney
bean.
Muscle Replicas
[0305] A large number of meat products comprise a high proportion
of skeletal muscle. Accordingly, the present invention provides a
composition derived from non-animal sources which replicates or
approximates key features of animal skeletal muscle. In another
aspect, the present invention provides a meat substitute product
that comprises a composition derived from non-animal sources which
replicates or approximates animal skeletal muscle. Such a
composition will be labeled herein as "muscle replica". In some
embodiments, the muscle replica and/or meat substitute product
comprising the muscle replica are partially derived from animal
sources. In some embodiments, the muscle replica and/or meat
substitute product comprising the muscle replica are entirely
derived from non-animal sources.
[0306] Many meat products comprise a high proportion of striated
skeletal muscle in which individual muscle fibers are organized
mainly in an isotropic fashion. Accordingly, in some embodiments
the muscle replica comprises fibers that are to some extent
organized isotropically. In some embodiments the fibers comprise a
protein component. In some embodiments, the fibers comprise about
1%, about 2%, about 5%, about 10%, about 15%, about 20%, about 30%,
about 40%, about 50%, about 60%, about 70%, about 80%, about 90%,
about 95%, about 99% or more of a protein component.
[0307] In some embodiments, the protein component comprises one or
more isolated, purified proteins. For example the one or more
isolated, purified protein can comprise the 8S globulin from Moong
bean seeds, or the albumin or globulin fraction of pea seeds. These
proteins provide examples of proteins with favorable properties for
constructing meat replicas because of their ability to form gels
with textures similar to animal muscle or fat tissue. Examples and
embodiments of the one or more isolated, purified proteins are
described herein. The list of potential candidates here is
essentially open and may include Rubisco, any major seed storage
proteins, proteins isolated from fungi, bacteria, archaea, viruses,
or genetically engineered microorganisms, or synthesized in vitro.
The proteins may be artificially designed to emulate physical
properties of animal muscle tissue. The proteins may be
artificially designed to emulate physical properties of animal
muscle tissue. In some embodiments, one or more isolated, purified
proteins accounts for about 0.1%, 0.2%, 0.5%, 1%, 2%, 3%, 4%, 5%,
6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%,
60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or more of the protein
component by weight.
[0308] Skeletal muscle of animals such as beef cattle typically
contain substantial quantities of glycogen, which can comprise on
the order of 1% of the mass of the muscle tissue at the time of
slaughter. After slaughter, a fraction of this glycogen continues
to be metabolized yielding products including lactic acid, which
contributes to lowering the pH of the muscle tissue, a desirable
quality in meat. Glycogen is a branched polymer of glucose linked
together by alpha (1->4) glycosidic bonds in linear chains, with
branch points comprising alpha (1->6) glycosidic bonds. Starches
from plants, particularly amylopectins are also branched polymers
of glucose linked together by alpha (1->4) glycosidic bonds in
linear chains, with branch points comprising alpha (1->6)
glycosidic bonds and can therefore be used as an analog of glycogen
in constructing meat replicas. Thus in some embodiments, the muscle
or meat replica includes a starch or pectin.
[0309] Additional components of animal muscle tissue include
sodium, potassium, calcium, magnesium, other metal ions, lactic
acid, other organic acids, free amino acids, peptides, nucleotides
and sulfur compounds. Thus in some embodiments, the muscle replica
can include sodium, potassium, calcium, magnesium, other metal
ions, lactic acid, other organic acids, free amino acids, peptides,
nucleotides and sulfur compounds. In some embodiments the
concentration of sodium, potassium, calcium, magnesium, other metal
ions, lactic acid, other organic acids, free amino acids, peptides,
nucleotides and/or sulfur compounds in the muscle replica or
consumable are within 10% of the concentrations found in a muscle
or meat being replicated.
[0310] In another aspect, the invention provides methods for making
a muscle replica. In some embodiments, the composition is formed
into asymmetric fibers prior to incorporation into the consumable.
In some embodiments these fibers replicate muscle fibers. In some
embodiments the fibers are spun fibers. In other embodiments the
fibers are extruded fibers. Accordingly, the present invention
provides for methods for producing asymmetric or spun protein
fibers. In some embodiments, the fibers are formed by extrusion of
the protein component through an extruder. Methods of extrusion are
well known in the art, and are described in U.S. Pat. No.
6,379,738, U.S. Pat. No. 3,693,533, US20120093994, which are herein
incorporated by reference.
[0311] In some embodiments extrusion can be conducted using an
MPF19 twin-screw extruder (APV Baker, Grand Rapids, Mich.) with a
cooling die. The cooling die can cool the extrudate prior to return
of the extrudate to atmospheric pressure, thus substantially
inhibiting expansion or puffing of the final product. In the MPF19
apparatus, dry feed and liquid can be added separately and mixed in
the barrel. Extrusion parameters can be, for example: screw speed
of 200 rpm, product temperature at the die of 150 C., feed rate of
23 g/min, and water-flow rate of 11 g/min. Product temperature can
be measured during extrusion by a thermocouple at the end of the
extrusion barrel. Observations can be made on color, opacity,
structure, and texture for each collected sample. Collected samples
can be optionally dried at room temperature overnight, then ground
to a fine powder (<60 mesh) using a Braun food grinder. The pH
of samples can be measured in duplicate using 10% (w/v) slurries of
powdered sample in distilled water.
[0312] Fat Replica
[0313] Animal fat is important for the experience of eating cooked
meat. Accordingly, the present invention provides a composition
derived from non-animal sources which recapitulates key features of
animal fat. In another aspect, the present invention provides a
meat substitute product that comprises a composition derived from
non-animal sources which recapitulates animal fat. Such a
composition will be labeled herein as a "fat replica". In some
embodiments, the fat replica and/or meat substitute product
comprising the fat replica are partially derived from animal
sources.
[0314] In some embodiments the meat substitute product has a fat
component. In some embodiments the fat content of the consumable is
1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, or 60%
fat. In some embodiments, the fat replica comprises a gel with
droplets of fat suspended therein. In some embodiments, the gel is
a soft, elastic gel comprising proteins and optionally
carbohydrates. In particular embodiments, the proteins used in the
gel are plant or microbial proteins. In some embodiments, the
proteins used in the fat replica might include Rubisco, any major
seed storage proteins, proteins isolated from fungi, bacteria,
archaea, viruses, or genetically engineered microorganisms, or
synthesized in vitro. The proteins may be artificially designed to
emulate physical properties of animal fat. The proteins may be
artificially designed to emulate physical properties of animal
fat.
[0315] The fat droplets used in some embodiments of the present
invention can be from a variety of sources. In some embodiments,
the sources are non-animal sources. In particular embodiments, the
sources are plant sources. Non-limiting examples of oils include
corn oil, olive oil, soy oil, peanut oil, walnut oil, almond oil,
sesame oil, cottonseed oil, rapeseed oil, canola oil, safflower
oil, sunflower oil, flax seed oil, algal oil, palm oil, palm kernel
oil, coconut oil, babassu oil, shea butter, mango butter, cocoa
butter, wheat germ oil, rice bran oil, oils produced by bacteria,
algae, archaea or fungi or genetically engineered bacteria, algae,
archaea or fungi, triglycerides, monoglycerides, diglycerides,
sphingosides, glycolipids, lecithin, lysolecithin, phosphatidic
acids, lysophosphatidic acids, oleic acid, palmitoleic acid,
palmitic acid, myristic acid, lauric acid, myristoleic acid,
caproic acid, capric acid, caprylic acid, pelargonic acid,
undecanoic acid, linoleic acid, 20:1 eicosanoic acid, arachidonic
acid, eicosapentanoic acid, docosohexanoic acid, 18:2 conjugated
linoleic acid, conjugated oleic acid, or esters of: oleic acid,
palmitoleic acid, palmitic acid, myristic acid, lauric acid,
myristoleic acid, caproic acid, capric acid, caprylic acid,
pelargonic acid, undecanoic acid, linoleic acid, 20:1 eicosanoic
acid, arachidonic acid, eicosapentanoic acid, docosohexanoic acid,
18:2 conjugated linoleic acid, or conjugated oleic acid, or
glycerol esters of oleic acid, palmitoleic acid, palmitic acid,
myristic acid, lauric acid, myristoleic acid, caproic acid, capric
acid, caprylic acid, pelargonic acid, undecanoic acid, linoleic
acid, 20:1 eicosanoic acid, arachidonic acid, eicosapentanoic acid,
docosohexanoic acid, 18:2 conjugated linoleic acid, or conjugated
oleic acid, or triglyceride derivatives of oleic acid, palmitoleic
acid, palmitic acid, myristic acid, lauric acid, myristoleic acid,
caproic acid, capric acid, caprylic acid, pelargonic acid,
undecanoic acid, linoleic acid, 20:1 eicosanoic acid, arachidonic
acid, eicosapentanoic acid, docosohexanoic acid, 18:2 conjugated
linoleic acid, or conjugated oleic acid.
[0316] In some embodiments, fat droplets are derived from pulp or
seed oil. In other embodiments, the source may be yeast or mold.
For instance, in one embodiment the fat droplets comprise
triglycerides derived from Mortierella isabellina.
[0317] In some embodiments plant oils are modified to resemble
animal fats. The plant oils can be modified with flavoring or other
agents to recapitulate the taste and smell of meat during and after
cooking. Accordingly, some aspects of the invention involve methods
for testing the qualitative similarity between the cooking
properties of animal fat and the cooking properties of plant oils
in the consumable.
[0318] In some embodiments, the fat replica comprises a protein
component comprising one or more isolated, purified proteins. The
purified proteins contribute to the taste and texture of the meat
replica. In some embodiments purified proteins can stabilize
emulsified fats. In some embodiments the purified proteins can form
gels upon denaturation or enzymatic crosslinking, which replicate
the appearance and texture of animal fat. Examples and embodiments
of the one or more isolated, purified proteins are described
herein. In particular embodiments, the one or more isolated
proteins comprise a protein isolated from the legume family of
plants. Non-limiting examples of legume plants are described
herein, although variations with other legumes are possible. In
some embodiments, the legume plant is a pea plant. In some
embodiments the isolated purified proteins stabilize emulsions. In
some embodiments the isolated purified proteins form gels upon
crosslinking or enzymatic crosslinking. In some embodiments, the
isolated, purified proteins comprise seed storage proteins. In some
embodiments, the isolated, purified proteins comprise albumin. In
some embodiments, the isolated, purified proteins comprise
globulin. In a particular embodiment, the isolated, purified
protein is a purified pea albumin protein. In another particular
embodiment, the isolated, purified protein is a purified pea
globulin protein. In another particular embodiment the isolate
purified protein is a Moong bean 8S globulin. In another particular
embodiment, the isolated, purified protein is an oleosin. In
another particular embodiment, the isolated, purified protein is a
caloleosin. In another particular embodiment, the isolated,
purified protein is Rubisco. In some embodiments, the protein
component comprises about 0.1%, 0.5%, 1%, 2%, 5%, 10%, 15%, 20%,
25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,
90% or more of the fat replica by dry weight or total weight. In
some embodiments, the protein component comprises about 0.1-5%,
about 0.5-10%, about 1-20%, about 5-30%, about 10-50%, about
20-70%, or about 30-90% or more of the fat replica by dry weight or
total weight. In some embodiments, the protein component comprises
a solution containing one or more isolated, purified proteins.
[0319] In some embodiments, the fat replica comprises cross-linking
enzymes that catalyze reactions leading to covalent crosslinks
between proteins. Cross-linking enzymes can be used to create or
stabilize the desired structure and texture of the adipose tissue
replica, to mimic the desired texture of an equivalent desired
animal fat. Non-limiting examples of cross-linking enzymes include,
e.g., transglutaminase, lysyl oxidases, or other amine oxidases
(e.g. Pichia pastoris lysyl oxidase). In some embodiments, the
cross-linking enzymes are isolated and purified from a non-animal
source, examples and embodiments of which are described herein. In
some embodiments, the fat replica comprises at least 0.0001%, or at
least 0.001%, or at least 0.01%, or at least 0.1%, or at least 1%
(wt/vol) of a cross-linking enzyme. In particular embodiments, the
cross-linking enzyme is transglutaminase.
[0320] In another aspect, the invention provides methods for making
a fat replica. In some embodiments, the fat droplets are suspended
in a gel. In some embodiments the present invention provides for
methods for producing droplets of fat suspended in the gel. The fat
can isolated and homogenized. For example an organic solvent
mixture can be used to help mix a lipid. The solvent can then be
removed. At this point the lipid can be frozen, lyophilized, or
stored. So in some aspects the invention provides for a method for
isolating and storing a lipid which has been selected to have
characteristics similar to animal fat. The lipid film or cake can
then be hydrated. The hydration can utilize agitation or
temperature changes. The hydration can occur in a precursor
solution to a gel. After hydration the lipid suspension can be
sonicated or extruded to further alter the properties of the lipid
in the solution.
[0321] In some embodiments, the fat replica is assembled to
approximate the organization adipose tissue in meat. In some
embodiments some or all of the components of the fat replica are
suspended in a gel. In various embodiments the gel can be a
proteinaceous gel, a hydrogel, an organogel, or a xerogel. In some
embodiments, the gel can be thickened to a desired consistency
using an agent based on polysaccharides or proteins. For example
fecula, arrowroot, cornstarch, katakuri starch, potato starch,
sago, tapioca, alginin, guar gum, locust bean gum, xanthan gum,
collagen, egg whites, furcellaran, gelatin, agar, carrageenan,
cellulose, methylcellulose, hydroxymethylcellulose, acadia gum,
konjac, starch, pectin, amylopectin or proteins derived from
legumes, grains, nuts, other seeds, leaves, algae, bacteria, of
fungi can be used alone or in combination to thicken the gel,
forming an architecture or structure for the consumable.
[0322] In particular embodiments, the fat replica is an emulsion
comprising a solution of one or more proteins and one or more fats
suspended therein as droplets. In some embodiments, the emulsion is
stabilized by one or more cross-linking enzymes into a gel. In more
particular embodiments, the one or more proteins in solution are
isolated, purified proteins. In yet more particular embodiments,
the isolated, purified proteins comprise a purified pea albumin
enriched fraction. In other more particular embodiments, the
isolated, purified proteins comprise a purified pea globulin
enriched fraction. In other more particular embodiments, the
isolated, purified proteins comprise a purified Moong bean 8S
globulin enriched fraction. In yet more particular embodiments, the
isolated, purified proteins comprise a Rubisco enriched fraction.
In other particular embodiments, the one or more fats are derived
from plant-based oils. In more particular embodiments, the one or
more fats are derived from one or more of: corn oil, olive oil, soy
oil, peanut oil, walnut oil, almond oil, sesame oil, cottonseed
oil, rapeseed oil, canola oil, safflower oil, sunflower oil, flax
seed oil, algal oil, palm oil, palm kernel oil, coconut oil,
babassu oil, shea butter, mango butter, cocoa butter, wheat germ
oil, rice bran oil, oils produced by bacteria, algae, archaea or
fungi or genetically engineered bacteria, algae, archaea or fungi,
triglycerides, monoglycerides, diglycerides, sphingosides,
glycolipids, lecithin, lysolecithin, phosphatidic acids,
lysophosphatidic acids, oleic acid, palmitoleic acid, palmitic
acid, myristic acid, lauric acid, myristoleic acid, caproic acid,
capric acid, caprylic acid, pelargonic acid, undecanoic acid,
linoleic acid, 20:1 eicosanoic acid, arachidonic acid,
eicosapentanoic acid, docosohexanoic acid, 18:2 conjugated linoleic
acid, conjugated oleic acid, or esters of: oleic acid, palmitoleic
acid, palmitic acid, myristic acid, lauric acid, myristoleic acid,
caproic acid, capric acid, caprylic acid, pelargonic acid,
undecanoic acid, linoleic acid, 20:1 eicosanoic acid, arachidonic
acid, eicosapentanoic acid, docosohexanoic acid, 18:2 conjugated
linoleic acid, or conjugated oleic acid, or glycerol esters of
oleic acid, palmitoleic acid, palmitic acid, myristic acid, lauric
acid, myristoleic acid, caproic acid, capric acid, caprylic acid,
pelargonic acid, undecanoic acid, linoleic acid, 20:1 eicosanoic
acid, arachidonic acid, eicosapentanoic acid, docosohexanoic acid,
18:2 conjugated linoleic acid, or conjugated oleic acid, or
triglyceride derivatives of oleic acid, palmitoleic acid, palmitic
acid, myristic acid, lauric acid, myristoleic acid, caproic acid,
capric acid, caprylic acid, pelargonic acid, undecanoic acid,
linoleic acid, 20:1 eicosanoic acid, arachidonic acid,
eicosapentanoic acid, docosohexanoic acid, 18:2 conjugated linoleic
acid, or conjugated oleic acid. In yet even more particular
embodiments, the one or more fats is a rice bran oil. In another
particular embodiment, the one or more fats is a canola oil. In
other particular embodiments, the cross-linking enzyme is
transglutaminase, lysyl oxidase, or other amine oxidase. In yet
even more particular embodiments, the cross-linking enzyme is
transglutaminase. In particular embodiments, the fat replica is a
high fat emulsion comprising a protein solution of purified pea
albumin emulsified with 40-80% rice bran oil, stabilized with
0.5-5% (wt/vol) transglutaminase into a gel. In particular
embodiments, the fat replica is a high fat emulsion comprising a
protein solution of partially-purified moong bean 8S globulin
emulsified with 40-80% rice bran oil, stabilized with 0.5-5%
(wt/vol) transglutaminase into a gel. In particular embodiments,
the fat replica is a high fat emulsion comprising a protein
solution of partially-purified moong bean 8S globulin emulsified
with 40-80% canola oil, stabilized with 0.5-5% (wt/vol)
transglutaminase into a gel. In particular embodiments, the fat
replica is a high fat emulsion comprising a protein solution of
purified pea albumin emulsified with 40-80% rice bran oil,
stabilized with 0.0001-1% (wt/vol) transglutaminase into a gel. In
particular embodiments, the fat replica is a high fat emulsion
comprising a protein solution of partially-purified moong bean 8S
globulin emulsified with 40-80% rice bran oil, stabilized with
0.0001-1% (wt/vol) transglutaminase into a gel. In particular
embodiments, the fat replica is a high fat emulsion comprising a
protein solution of partially-purified moong bean 8S globulin
emulsified with 40-80% canola oil, stabilized with 0.0001-1%
(wt/vol) transglutaminase into a gel.
[0323] Connective Tissue Replica
[0324] Animal connective tissue provides key textural features that
are an important component of the experience of eating meat.
Accordingly, the present invention provides a composition derived
from non-animal sources which recapitulates key features of animal
connective tissue. In another aspect, the present invention
provides a meat substitute product that comprises a composition
derived from non-animal sources which recapitulates important
textural and visual features of animal connective tissue. Such a
composition will be labeled herein as "connective tissue replica".
In some embodiments, the connective tissue replica and/or meat
substitute product comprising the connective tissue replica are
partially derived from animal sources.
[0325] Animal connective tissue can generally be divided into
fascia-type and cartilage-type tissue. Fascia-type tissue is highly
fibrous, resistant against extension (has high elastic modulus),
and has a high protein content, a moderate water content (ca. 50%),
and low-to-none fat and polysaccharide content. Accordingly, the
present invention provides a connective tissue replica that
recapitulates key features of fascia type tissue. In some
embodiments, the connective tissue replica comprises about 50%
protein by total weight, about 50% by liquid weight, and has a low
fat and polysaccharide component.
[0326] The protein content of most fascia-type connective tissue is
comprised mainly of collagen. Collagen is characterized by a high
fraction of proline and alanine, and also is assembled into
characteristic elongated fibrils or rod-like, flexible structures.
Prolamins are one family of proteins found in non-animal sources,
such as plant sources. Prolamins are highly abundant in plants and
are similar in amino acid composition to collagen. Among proteins
we tested for this purpose, prolamins were particularly favorable
because of their low cost and their ability to readily form fibers
or sheets when spun or extruded. Non-limiting examples of prolamin
family proteins include, e.g., zein (found in corn), these include
hordein from barley, gliadin from wheat, secalin, extensins from
rye, kafirin from sorghum, avenin from oats. In fascia-type
connective tissue, the prolamin family of proteins, individually or
combinations thereof, demonstrates suitability for the protein
component because they are highly abundant, similar in global amino
acid composition to collagen (high fraction of proline and
alanine), and amenable to processing into films and fibers. In
addition to zein (found in corn), these include hordein from
barley, gliadin from wheat, secalin, extensins from rye, kafirin
from sorghum, avenin from oats. Other proteins may be necessary to
supplement prolamins in order to achieve targets specifications for
physicochemical and nutritional properties. The list of potential
candidates here is essentially open and may include Rubisco, any
major seed storage proteins, proteins isolated from fungi,
bacteria, archaea, viruses, or genetically engineered
microorganisms, or synthesized in vitro. The proteins may be
artificially designed to emulate physical properties of animal
connective tissue. animal-derived or recombinant collagen,
extensins (hydroxyproline-rich glycoproteins abundant in cell walls
e.g. Arabidopsis thaliana, monomers of which are "collagen-like"
rod-like flexible molecules). The proteins may be artificially
designed to emulate physical properties of animal connective
tissue.
[0327] Methods for forming fascia-type connective tissue will be as
those practiced in the art with a bias towards methods producing
fibrous or fibrous-like structures by biological, chemical, or
physical means, individually or in combination, serially or in
parallel, before final forming. These methods may include extrusion
or spinning.
[0328] Cartilage-type tissue is macroscopically homogenous,
resistant against compression, has higher water content (up to
80%), lower protein (collagen) content, and higher polysaccharide
(proteoglycans) contents (ca. 10% each).
[0329] Compositionally, cartilage-type connective tissue will be
very similar to fascia-type tissue with the relative ratios of each
adjusted to more closely mimic `meat` connective tissue.
[0330] Methods for forming cartilage-type connective tissue will be
similar to those for fascia-type connective tissue, but with a bias
towards methods producing isotropically homogenous structures.
[0331] The fat can be suspended in a gel. In some embodiments the
present invention provides for methods for producing droplets of
fat suspended in the proteinaceous gel. The fat can be isolated
from plant tissues and emulsified. The emulsification can utilize
high-speed blending, homogenization, agitation or temperature
changes. The lipid suspension can be sonicated or extruded to
further alter the properties of the lipid in the solution. At this
point, in some embodiments other components of the consumable are
added to the solution followed by a gelling agent. In some
embodiments crosslinking agents (e.g. transglutaminase or lysyl
oxidase) are added to bind the components of the consumable. In
other embodiments the gelling agent is added and the lipid/gel
suspension is later combined with additional components of the
consumable. In fascia-type connective tissue, the prolamin family
of proteins, individually or combinations thereof, demonstrates
suitability for the protein component because they are highly
abundant, similar in global amino acid composition to collagen
(high fraction of proline and alanine), and amenable to processing
into films. In addition to zein (found in corn), these include
hordein from barley, gliadin from wheat, secalin, extensions from
rye, kafirin from sorghum, avenin from oats. Other proteins may be
necessary to supplement prolamins in order to achieve targets
specifications for physicochemical and nutritional properties. The
list of potential candidates here is essentially open and may
include any major seed storage proteins, animal-derived or
recombinant collagen, extensins (hydroxyproline-rich glycoproteins
abundant in cell walls e.g. Arabidopsis thaliana, monomers of which
are "collagen-like" rod-like flexible molecules).
[0332] In some embodiments some or all of the components of the
consumable are suspended in a gel. In various embodiments the gel
can be a hydrogel, an organogel, or a xerogel, The gel can be made
thick using an agent based on polysaccharides or proteins. For
example fecula, arrowroot, cornstarch, katakuri starch, potato
starch, sago, tapioca, alginin, guar gum, locust bean gum, xanthan
gum, collagen, egg whites, furcellaran, gelatin, agar, carrageenan,
cellulose, methylcellulose, hydroxymethylcellulose, acadia gum,
konjac, starch, pectin, amylopectin or proteins derived from
legumes, grains, nuts, other seeds, leaves, algae, bacteria, of
fungi can be used alone or in combination to thicken the gel,
forming an architecture or structure for the consumable. Enzymes
that catalyze reactions leading to covalent crosslinks between
proteins can also be used alone or in combination to form an
architecture or structure for the consumable. For example
transclutaminase, lysyl oxidases, or other amine oxidases (e.g.
Pichia pastoris lysyl oxidase (PPLO)) can be used alone or in
combination to form an architecture or structure for the
consumable. In some embodiments multiple gels with different
components are combined to form the consumable. For example a gel
containing a plant-based protein can be associated with a gel
containing a plant-based fat. In some embodiments fibers or stings
of proteins are oriented parallel to one another and then held in
place by the application of a gel containing plant based fats.
[0333] The compositions of the invention can be puffed or expanded
by heating, such as frying, baking, microwave heating, heating in a
forced air system, heating in an air tunnel, and the like,
according to methods well known in the art.
[0334] In some embodiments multiple gels with different components
are combined to form the consumable. For example a gel containing a
plant-based protein can be associated with a gel containing a
plant-based fat. In some embodiments fibers or strings of proteins
are oriented parallel to one another and then held in place by the
application of a gel containing plant based fats.
[0335] In some embodiments the meat replica contains no animal
products, less than 1% wheat gluten, no methylcellulose, no
carrageenan, no caramel color and no Konjac flour, no gum Arabic,
and no acacia gum.
[0336] In some embodiments the meat replica contains no animal
products, no wheat gluten, no methylcellulose, no carrageenan, no
caramel color and no Konjac flour, no gum Arabic, and no acacia
gum.
[0337] In some embodiments the meat replica contains no animal
products, no soy protein isolate, no wheat gluten, no
methylcellulose, no carrageenan, no caramel color and no Konjac
flour, no gum Arabic, and no acacia gum.
[0338] In some embodiments the meat replica contains no animal
products, no soy protein concentrate, no wheat gluten, no
methylcellulose, no carrageenan, no caramel color and no Konjac
flour, no gum Arabic, and no acacia gum.
[0339] In some embodiments the meat replica contains no animal
products, no soy protein, no wheat gluten, no methylcellulose, no
carrageenan, no caramel color and no Konjac flour, no gum Arabic,
and no acacia gum.
[0340] In some embodiments the meat replica contains no animal
products, no tofu, no wheat gluten, no methylcellulose, no
carrageenan, no caramel color and no Konjac flour, no gum Arabic,
and no acacia gum.
[0341] In some embodiments the meat replica contains no animal
products, no tofu, and no wheat gluten.
[0342] In some embodiments the meat replica contains no animal
products, no soy protein, and no wheat gluten.
[0343] In some embodiments the meat replica contains no
methylcellulose, no carrageenan, no caramel color, no Konjac flour,
no gum Arabic, and no acacia gum.
[0344] In some embodiments the meat replica contains no animal
products and less than 5% carbohydrates.
[0345] In some embodiments the meat replica contains no animal
products, no soy protein, no wheat gluten, no methylcellulose, no
carrageenan, no caramel color and no Konjac flour, no gum Arabic,
and no acacia gum and less than 5% carbohydrates.
[0346] In some embodiments the meat replica contains no animal
products, and less than 1% cellulose.
[0347] In some embodiments the meat replica contains no animal
products, and less than 5% insoluble carbohydrates.
[0348] In some embodiments the meat replica contains no animal
products, no soy protein, and less than 1% cellulose.
[0349] In some embodiments the meat replica contains no animal
products, no soy protein, and less than 5% insoluble
carbohydrates.
[0350] In some embodiments the meat replica contains no animal
products, no wheat gluten, and less than 1% cellulose.
[0351] In some embodiments the meat replica contains no animal
products, no wheat gluten, and less than 5% insoluble
carbohydrates.
[0352] The percentage of different components may also be
controlled. For example non-animal-based substitutes for muscle,
fat tissue, connective tissue, and blood components can be combined
in different ratios and physical organizations to best approximate
the look and feel of meat. The various can also components can be
arranged to insure consistency between bites of the consumable. The
components can be arranged to insure that no waste is generated
from the consumable. For example, while a traditional cut of meat
may have portions that are not typically eaten, a meat replicate
can improve upon meat by not including these inedible portions.
Such an improvement allows for all of the product made or shipped
to be consumed, which cuts down on waste and shipping costs.
Alternatively, a meat replica may include inedible portions to
mimic the experience of meat consumption. Such portions can include
bone, cartilage, connective tissue, or other materials commonly
referred to as gristle, or materials included simulating these
components. In some embodiments the consumable may contain
simulated inedible portions of meat products which are designed to
serve secondary functions. For example a simulated bone can be
designed to disperse heat during cooking, making the cooking of the
consumable faster or more uniform than meat. In other embodiments a
simulated bone may also serve to keep the consumable at a constant
temperature during shipping. In other embodiments, the simulated
inedible portions may be biodegradable.
[0353] In some embodiments the meat substitute compositions
contains no animal protein, comprising between 10-30% protein,
between 5-80% water, between 5-70% fat, comprising one or more
isolated purified proteins. In particular embodiments, the meat
substitute compositions comprise transglutaminase.
[0354] In some embodiments the consumable contains components to
replicate the components of meat. The main component of meat is
typically skeletal muscle. Skeletal muscle typically consists of
roughly 75 percent water, 19 percent protein, 2.5 percent
intramuscular fat, 1.2 percent carbohydrates and 2.3 percent other
soluble non-protein substances. These include organic acids, sulfur
compounds, nitrogenous compounds, such as amino acids and
nucleotides, and inorganic substances such as minerals.
Accordingly, some embodiments of the present invention provide for
replicating approximations of this composition for the consumable.
F or example, in some embodiments the consumable is a plant-based
meat replica can comprise roughly 75% water, 19% protein, 2.5% fat,
1.2% carbohydrates; and 2.3 percent other soluble non-protein
substances. In some embodiments the consumable is a plant-based
meat replica comprising between 60-90% water, 10-30% protein, 1-20%
fat, 0.1-5% carbohydrates; and 1-10 percent other soluble
non-protein substances. In some embodiments the consumable is a
plant-based meat replica comprising between 60-90% water, 5-10%
protein, 1-20% fat, 0.1-5% carbohydrates; and 1-10 percent other
soluble non-protein substances. In some embodiments the consumable
is a plant-based meat replica comprising between 0-50% water, 5-30%
protein, 20-80%% fat, 0.1-5% carbohydrates; and 1-10 percent other
soluble non-protein substances. In some embodiments, the replica
contains between 0.01% and 5% by weight of a heme protein. In some
embodiments, the replica contains between 0.01% and 5% by weight of
leghemoglobin. Some meat also contains myoglobin, a heme protein,
which accounts for most of the red color and iron content of some
meat. In some embodiments, the replica contains between 0.01% and
5% by weight of a heme protein. In some embodiments, the replica
contains between 0.01% and 5% by weight of leghemoglobin. It is
understood that these percentages can vary in meat and the meat
replicas can be produced to approximate the natural variation in
meat. Additionally, in some instances, the present invention
provides for improved meat replicas, which comprise these
components in typically unnatural percentages. For example a meat
replica can be produced with a higher than typical average fat
content. The percentages of these components may also be altered to
increase other desirable properties.
[0355] In some instances a meat replica is designed so that, when
cooked, the percentages of components are similar to cooked meat.
So, in some embodiments, the uncooked consumable has different
percentages of components than uncooked meat, but when cooked the
consumable is similar to cooked meat. For example, a meat replica
may be made with a higher than typical water content for raw meat,
but when cooked in a microwave the resulting product has
percentages of components similar to meat cooked over a fire.
[0356] In some embodiments the consumable is a meat replica with a
lower that typical water content for meat. In some embodiments the
inventions provides for methods for hydrating a meat replica to
cause the meat replica to have a content similar to meat. For
example a meat replica with a water content that would be low for
meat, for example 1%, 10%, 20%, 30%, 40% or 50% water, is hydrated
to roughly 75% water. Once hydrated, in some embodiments, the meat
replica is then cooked for human consumption.
[0357] The consumable can have a protein component. In some
embodiments the protein content of the consumable is 10%, 20%, 30%,
or 40%. In some embodiments the protein content of the consumable
is similar to meat. In some embodiments the protein content in the
consumable is greater than that of meat. In some embodiments the
consumable has less protein than meat.
[0358] The protein in the consumable can come from a variety or
combination of sources. Non-animal sources can provide some or all
of the protein in the consumable. Non-animal sources can include
vegetables, fruits, nuts, grains, algae, bacteria, or fungi. The
protein can be isolated or concentrated from one or more of these
sources. In some embodiments the consumable is a meat replica
comprising protein only obtained from non-animal sources.
[0359] In some embodiments protein is formed into asymmetric fibers
for incorporation into the consumable. In some embodiments these
fibers replicate muscle fibers. In some embodiments the protein are
spun fibers. Accordingly, the present invention provides for
methods for producing asymmetric or spun protein fibers. In some
embodiments the consumable contains a protein or proteins that have
all of the amino acids found in proteins that are essential for
human nutrition. In some embodiments the proteins added to the
consumable are supplemented with amino acids.
[0360] Indicators of Cooking Meat
[0361] The release of odorants upon cooking is an important aspect
of meat consumption. In some embodiments, the consumable is a meat
replica entirely composed of non-animal products that when cooked
generates an aroma recognizable by humans as typical of cooking
beef. In some embodiments, the consumable when cooked generates an
aroma recognizable by humans as typical of cooking pork. In some
embodiments, the consumable is a meat replica entirely composed of
non-animal products that when cooked generates an aroma
recognizable by humans as typical of cooking bacon. In some
embodiments, the consumable is a meat replica entirely composed of
non-animal products that when cooked generates an aroma
recognizable by humans as typical of cooking chicken. In some
embodiments, the consumable is a meat replica entirely composed of
non-animal products that when cooked generates an aroma
recognizable by humans as typical of cooking lamb. In some
embodiments, the consumable is a meat replica entirely composed of
non-animal products that when cooked generates an aroma
recognizable by humans as typical of cooking fish. In some
embodiments, the consumable is a meat replica entirely composed of
non-animal products that when cooked generates an aroma
recognizable by humans as typical of cooking turkey. In some
embodiments the consumable is a meat replica principally or
entirely composed of ingredients derived from non-animal sources,
with an odorant that is released upon cooking. In some embodiments
the consumable is a meat replica principally or entirely composed
of ingredients derived from non-animal sources, with an odorant
that is produced by chemical reactions that take place upon
cooking. In some embodiments the consumable is a meat replica
principally or entirely composed of ingredients derived from
non-animal sources, containing mixtures of proteins, peptides,
amino acids, nucleotides, sugars and polysaccharides and fats in
combinations and spatial arrangements that enable these compounds
to undergo chemical reactions during cooking to produce odorants
and flavor-producing compounds. In some embodiments the consumable
is a meat replica principally or entirely composed of ingredients
derived from non-animal sources, with a volatile or labile odorant
that is released upon cooking. In some embodiments the consumable
is a method for preparing a meat replica where meat replicas
principally or entirely composed of ingredients derived from
non-animal sources are heated to release a volatile or labile
odorant.
[0362] Odorants released during cooking of meat are generated by
reactions that can involve as reactants fats, protein, amino acids,
peptides, nucleotides, organic acids, sulfur compounds, sugars and
other carbohydrates. In some embodiments the odorants that combine
during the cooking of meat are identified and located near one
another in the consumable, such that upon cooking of the consumable
the odorants combine. So, in some embodiments, the characteristic
flavor and fragrance components are produced during the cooking
process by chemical reactions involving amino acids, fats and
sugars found in plants as well as meat. So, in some embodiments,
the characteristic flavor and fragrance components are mostly
produced during the cooking process by chemical reactions involving
one or more amino acids, fats, peptides, nucleotides, organic
acids, sulfur compounds, sugars and other carbohydrates found in
plants as well as meat.
[0363] Some reactions that generate odorants released during
cooking of meat can be catalyzed by iron, in particular the heme
iron of myoglobin. Thus in some embodiments, some of the
characteristic flavor and fragrance components are produced during
the cooking process by chemical reactions catalyzed by iron. In
some embodiments, some of the characteristic flavor and fragrance
components are produced during the cooking process by chemical
reactions catalyzed by heme. In some embodiments, some of the
characteristic flavor and fragrance components are produced during
the cooking process by chemical reactions catalyzed by the heme
iron in leghemoglobin. In some embodiments, some of the
characteristic flavor and fragrance components are produced during
the cooking process by chemical reactions catalyzed by the heme
iron in a heme protein.
[0364] Evidence that the presence of leghemoglobin contributes
favorably to aroma of meat replicas: A muscle replica comprising
pea flour, sunflower oil, and glucose was heated for 10 minutes at
140 C in the presence of either reduced leghemoglobin (LHb) or a
mixture of iron (Fe3+), sodium and EDTA (EFS) in sealed containers
carrying solid phase microextraction (SPME) fibers. These fibers
contain polydimethylsiloxane (PDMS) which adsorbs volatile
compounds for analysis by GC-MS. Analysis of GC-MS data from
multiple replicas reveal consistent differences between the LHb and
EFS samples. Non-limiting examples of compounds found exclusively
or more abundantly in the LHb samples are: 2-octanone, 2-methyl
furan, which are often associated with the aroma of cooked meat,
and many other unidentified compounds.
[0365] Color Indicators
[0366] The color of meat is an important part the experience of
cooking and eating meat. For instance, cuts of beef are of a
characteristic red color in a raw state and gradually transition to
a brown color during cooking. As another example, white meats such
as chicken or pork have a characteristic pink color in their raw
state and gradually transition to a white or brownish color during
cooking. The amount of the color transition is used to indicate the
cooking progression of beef and titrate the cooking time and
temperature to produce the desired state of done-ness. In some
aspects, the invention provides a non-meat based meat substitute
product that provides a visual indicator of cooking progression. In
some embodiments, the visual indicator is a color indicator that
undergoes a color transition during cooking. In particular
embodiments, the color indicator recapitulates the color transition
of a cut of meat as the meat progresses from a raw to a cooked
state. In more particular embodiments, the color indicator colors
the meat substitute product a red color before cooking to indicate
a raw state and causes the meat substitute product to transition to
a brown color during cooking progression. In other particular
embodiments, the color indicator colors the meat substitute product
a pink color before cooking to indicate a raw state and causes the
meat substitute product to transition to a white or brown color
during cooking progression.
[0367] The main determinant of the nutritional definition of the
color of meat is the concentration of iron carrying proteins in the
meat. In the skeletal muscle component of meat products, one of the
main iron-carrying proteins is myoglobin. It is estimated that the
white meat of chicken has under 0.05%; pork and veal have 0.1-0.3%;
young beef has 0.4-1.0%; and old beef has 1.5-2.0%. So, in some
embodiments, the consumable is a meat replica which comprises an
iron-carrying protein. In some embodiments, the meat replica
comprises about 0.05%, about 0.1%, about 0.2%, about 0.3%, about
0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%,
about 1%, about 1.1%, about 1.2%, about 1.3%, about 1.4%, about
1.5%, about 1.6%, about 1.7%, about 1.8%, about 1.9%, about 2%, or
more than about 2% of an iron-carrying protein by dry weight or
total weight. In some cases, the iron carrying protein has been
isolated and purified from a source. In other cases, the iron
carrying protein has not been isolated and purified. In some cases,
the source of the iron-carrying protein is an animal source, or a
non-animal source such as a plant, fungus, or genetically modified
organisms such as, e.g., bacteria or yeast. In some cases, the
iron-carrying protein is myoglobin. In some embodiments the
consumable is a plant based meat replica that has animal myoglobin
added. So, for example a replica of young beef can have about
0.4-1% myoglobin. In some cases, the iron-carrying protein is
leghemoglobin. In some embodiments the consumable is a plant based
meat replica that has leghemoglobin added. So, for example a
replica of young beef can have about 0.4-1% leghemoglobin. In some
cases, the iron-carrying protein is a cytochrome. In some
embodiments the consumable is a plant based meat replica that has a
cytochrome added. So, for example a replica of young beef can have
about 0.4-1% of a cytochrome.
[0368] Another example of iron-carrying proteins is hemoglobin, the
iron-containing oxygen-binding protein in the red blood cells of
vertebrates. Hemoglobin is similar in color to myoglobin. In some
embodiments the invention provides methods of saving and recycling
blood from animal farming to supplement the color of a consumable.
For example blood is saved from a slaughter house, hemoglobin from
the blood is used to enhance the color of a consumable. In some
aspects the consumable is a plant-based meat replica containing
hemoglobin.
[0369] Additional iron containing proteins exist in nature. In some
embodiments the consumable comprises an iron containing protein
that is not myoglobin. In some embodiments the consumable does not
contain myoglobin. In some embodiments the consumable does not
contain hemoglobin. In some embodiments the consumable is a meat
replica that comprises an iron containing protein other than
myoglobin or hemoglobin.
[0370] Examples of iron containing proteins include hemoglobin,
myoglobin, neuroglobin, cytoglobin, leghemoglobin, non-symbiotic
hemoglobin, Hell's gate globin I, bacterial hemoglobins, ciliate
myoglobins, flavohemoglobins. In various embodiments these iron
containing proteins are added to the consumable to alter the visual
characteristics or iron content of the consumable. In some
embodiments the consumable comprises a hemoprotein (e.g.
hemoglobin, myoglobin, neuroglobin, cytoglobin, leghemoglobin,
non-symbiotic hemoglobin, Hell's gate globin I, bacterial
hemoglobins, ciliate myoglobins, flavohemoglobins).
[0371] Leghemoglobin, similar in structure and physical properties
to myoglobin, is readily available as an unused by-product of
commodity legume crops (eg., soybean, pea). The leghemoglobin in
the roots of these crops in the US exceeds the myoglobin content of
all the red meat consumed in the US. In some embodiments the
consumable is a meat replica principally or entirely composed of
ingredients derived from non-animal sources, including a muscle
tissue replica, an adipose tissue replica, a connective tissue
replica, and leghemoglobin. In some embodiments the consumable is a
meat replica principally or entirely composed of ingredients
derived from non-animal sources, containing a heme protein. In some
embodiments the consumable is a meat replica principally or
entirely composed of ingredients derived from non-animal sources,
containing a leghemoglobin. In some embodiments the consumable is a
meat replica principally or entirely composed of ingredients
derived from non-animal sources, containing a member of the globin
protein family. In some embodiments the consumable is a meat
replica principally or entirely composed of ingredients derived
from non-animal sources, with a high iron content from a heme
protein. In some embodiments the iron content is similar to meat.
In some embodiments the consumable has the distinctive red color of
meat, such color provided by leghemoglobin.
[0372] Leghemoglobin is, in some embodiments, used as an indicator
that the consumable is finished cooking. So, one embodiment of the
invention is a method for cooking a consumable comprising detecting
leghemoglobin which has migrated from the interior of the
consumable to the surface when the product is cooked. Another
embodiment of the invention is a method for cooking a consumable
comprising detecting the change in color of from red to brown when
the product is cooked.
[0373] A heme protein is, in some embodiments, used as an indicator
that the consumable is finished cooking. So, one embodiment of the
invention is a method for cooking a consumable comprising detecting
leghemoglobin which has migrated from the interior of the
consumable to the surface when the product is cooked. Another
embodiment of the invention is a method for cooking a consumable
comprising detecting the change in color of from red to brown when
the product is cooked.
[0374] A heme protein from the group of: Hemoglobin, myoglobin,
neuroglobin, cytoglobin, leghemoglobin, non-symbiotic hemoglobin,
Hell's gate globin I, bacterial hemoglobins, ciliate myoglobins,
flavohemoglobins, is, in some embodiments, used as an indicator
that the consumable is finished cooking. So, one embodiment of the
invention is a method for cooking a consumable comprising detecting
leghemoglobin which has migrated from the interior of the
consumable to the surface when the product is cooked. Another
embodiment of the invention is a method for cooking a consumable
comprising detecting the change in color of from red to brown when
the product is cooked.
Food Products Comprising Isolated, Purified Leghemoglobin
[0375] In some embodiments leghemoglobin is added to meat to
enhance the properties of meat. For example, a leghemoglobin
containing solution can be injected into raw or cooked meat. In
another example a leghemoglobin solution is dripped over meat or a
consumable of the invention to enhance appearance. In one
embodiment advertising, photography, or videography of food
products such as meat or a meat substitute is enhanced with
leghemoglobin.
Sources of Leghemoglobin
[0376] In some embodiments the present invention provides methods
for obtaining leghemoglobin from plants. Leghemoglobin can be
obtained from a variety of plants. Various legumes species and
their varieties, for example, Soybean, Fava bean, Lima bean,
Cowpeas, English peas, Yellow peas, Lupine, Kidney bean, Garbanzo
beans, Peanut, Alfalfa, Vetch hay, Clover, Lespedeza and Pinto
bean, contain nitrogen-fixing root nodules in which leghemoglobin
has a key role in controlling oxygen concentrations (for example
root nodules from a pea plant, FIG. 1). FIG. 2 shows 100 mls of
leghemoglobin solution isolated from 30 grams of pea root nodules.
Leghemoglobins from different species are homologs and have similar
color properties (FIG. 3). In FIG. 3, panel A shows an SDS_PAGE
gels of lysed root-nodules of three legume plant species (1) Fava
bean (2) English Pea (3) Soybean. Arrows mark respective
leghemoglobins. Note that leghemoglobin is the most abundant
soluble protein in each lysate. Panel B shows the similarity of
UV-VIS spectral profile of leghemoglobins from two different plant
species (Favabean and Soybean). We purified leghemoglobin from fava
bean (green curve) and Soybean (red curve) root nodules using the
protocol described elsewhere in the specification. UV-VIS spectra
of both proteins shows that the heme iron is in the reduced (+2)
state. Note that they are almost perfectly superimposed, consistent
with their visually identical red color. The heme iron in the
respective leghemoglobins was reduced to the +2 oxidation state by
incubating Fava bean and Soybean leghemoglobin with 10 mM sodium
hydrosulfite in 20 mM potassium-phosphate pH 7.4, 100 mM sodium
chloride buffer. Sodium hydrosulfite was then removed from the
leghemoglobin solution using gel-exclusion chromatography. Inset
shows a zoom-in of UV-VIS spectra in 450 nm to 700 nm region. Some
plant species express several leghemoglobin isoforms (for example
soybean has four leghemoglobin isoforms). Minor variations in
precise amino acid sequence can modify overall charge of the
protein at a particular pH and can modify precise structural
conformation of iron containing heme group in leghemoglobin.
Differences in structural conformation of heme group of different
leghemoglobins can influence oxidation and reduction rates of the
heme iron. These differences may contribute to color and flavor
generation properties of different leghemoglobins.
[0377] Leghemoglobin has a virtually identical absorbance spectrum
and visual appearance to myoglobin from animal muscle. FIG. 4 shows
a comparison of reduced (heme iron 2+) and oxidized (heme iron 3+)
soybean leghemoglobin (FIG. 4 panel A) and equine heart muscle
myoglobin (FIG. 4 panel B) showing similarity of UV-VIS absorption
profiles of two proteins. We purified soybean leghemoglobin from
soybean root-nodules using here described protocol. Purified equine
myoglobin was purchased from SigmaAldrich. Soybean leghemoglobin
(FIG. 4 panel A) and equine myoglobin (FIG. 4 panel B) were reduced
with 1 mm sodium hydrosulfite. Shown are UV-VIS absorption spectra
of heme Fe3+ (blue line) and heme Fe2+ (red line) of soybean
leghemoglobin (FIG. 4 panel A) and equine myoglobin (FIG. 4 panel
B). Insets show a zoom-in of UV-VIS spectra in 450 nm to 700 nm
region. (FIG. 4 panel C) Images of 10 ul liquid droplet of a 40
mg/ml solution of soybean leghemoglobin in the heme-Fe3+ state
(left droplet) showing characteristic rusty red color and a 40
mg/ml solution of soybean leghemoglobin solution in the heme-Fe2+
state (right droplet) showing characteristic red color of and
(right image) corresponding samples of equine myoglobin.
[0378] In other embodiments, leghemoglobin can be sourced from
non-plant sources, such as from organisms such as bacteria or yeast
which have been genetically modified to express high levels of
leghemoglobin.
[0379] The oxidation state of the iron ion in leghemoglobin is
important for its color. Leghemoglobin with the heme iron in the +2
oxidation state appears vivid red in color, while leghemoglobin
with the heme iron in the +3 oxidation state appears brownish red.
Thus, in using leghemoglobin as a source of red color in a meat
replica, it is desirable to reduce the heme iron from the +3 state
to the +2 state. Heme iron in leghemoglobin can be switched from
oxidized (+3) state to reduced (+2) state with reducing reagents.
Examples of successful reduction of leghemoglobin heme iron with
sodium hydrosulfite and titanium citrate are illustrated in FIG. 5.
In FIG. 5 the UV-VIS spectrogram of purified soybean leghemoglobin
in which the heme iron is in the oxidized (+3) state is represented
by the blue curves in each panel. The red curves in each panel
represent the UV-VIS spectra of the same leghemoglobin species
after reduction to the (+2) state (red lines) by addition of (Panel
A) 1 mM sodium hydrosulfite or (Panel B) 0.24% (wt/v) titanium
citrate in 20 mM potassium phosphate pH 7.3, 100 mM sodium chloride
buffer. The Insets show a zoom-in of UV-VIS spectra in 450-700 nm
region. For this example, leghemoglobin was purified from soybean
root nodules using 60/90% ammonium sulfate fractionation and
exchanged into 20 mM potassium phosphate pH 7.4, 100 mM sodium
chloride buffer. Sodium hydrosulfite stock solution was prepared by
dissolving 100 mM sodium hydrosulfite in 1 mM sodium hydroxide in
water. Titanium citrate stock solution was prepared from 20% (wt/v)
Ti-chloride in hydrochloric acid by mixing it with 0.2M sodium
citrate (1:10 v/v). pH was adjusted using sodium carbonate to pH
7.0.
[0380] Leghemoglobin can be purified from legume root nodules, such
as the root nodules of peas or soybeans (FIG. 1 shows Leghemoglobin
isolated from pea root nodules). Root nodules from soy beans were
thoroughly cleaned to remove soil and extraneous root tissues prior
to root nodule lysis in 20 mM potassium phosphate pH 7.4, 100 mM
sodium chloride, 1 mm EDTA and 1 mM ascorbic acid. Root nodules
were lysed by grinding root-nodules using a Vitamix blender. For
some samples Polyvinylpyrrolidone polymer was added at 30% wt/v to
aid in removal of plant phenolic small molecules that mediate
oxidation of leghemoglobin heme-iron. Root nodule lysate was
fractionated using ammonium sulfate in two steps, first ammonium
sulfate was added to 60% wt/v. Pellet was discarded and supernatant
brought to 90% wt/v. ammonium sulfate. Leghemoglobin was collected
as a precipitated pellet in 90% ammonium sulfate fraction. Ammonium
sulfate precipitated leghemoglobin was resuspended in 20 mM
potassium phosphate, 1 mM EDTA, 50 mM sodium chloride and ammonium
sulfate was removed using dialysis or size-exclusion chromatography
in the same buffer. In some instances this was the last
purification step, while in other instances leghemoglobin was
further purified using anion-exchange chromatography (FFQ GE
Healthcare), which was sometimes followed by size-exclusion
chromatography (Sephacryl S-100, GE Healthcare). Soybean
leghemoglobin from 90% ammonium sulfate pellet was loaded on anion
exchange columns (FFQ or DEAE, GE Healthcare) in different buffers
(20 mM potassium phosphate pH 7.4, containing 0 to 100 mM sodium
chloride, 20 mM Tris pH 8 containing 0 to 100 mM sodium chloride,
20 mM sodium borax pH 9.8, 20 mM sodium chloride, 20 mM sodium
carbonate pH 9, 20 mM sodium chloride) and purified either in
flow-through or using sodium chloride (0-1M salt gradient). An
example of the leghemoglobin purification flow from soybean root
nodules is represented in FIG. 6. The figure shows SDS-PAGE
fractionation of different soybean leghemoglobin purification steps
(Lane 1) Soybean root-nodule lysate; (Lane 2) Soybean root-nodule
lysate purified by 60/90% (wt/v) ammonium sulfate fractionation.
Shown is the protein content of 90% ammonium sulfate fractionated
protein pellet resuspended in 20 mm potassium phosphate pH 7.4, 100
mM sodium chloride, 1 mM EDTA buffer; Proteins from 90% ammonium
sulfate pellet were further purified by anion-exchange
chromatography (FFQ GE Healthcare) in 20 mM potassium phosphate ph
7.4, 100 mM sodium chloride. Leghemoglobin collected in the
flowthrough is shown in Lane 3. Anion-exchange flowthrough was
fractionated using size-exclusion chromatography (Sephacryl S-100
GE Healthcare) and resulting leghemoglobin fraction is shown in
Lane 4. Leghemoglobin content at different purification steps was
determined by determining the fraction of leghemoglobin band on
SDS-PAGE gel in a respective sample using ImageDoc analysis
software (BioRad). Purity (partial abundance) of leghemoglobin at
respective steps in the purification steps was: lysate: 32.7% (lane
1), 60/90% (wt/v) ammonium sulfate fractionation 78% (lane 2),
anion-exchange chromatography .about.83% (lane 3), and
size-exclusion chromatography to .about.95% (lane 4).
[0381] Leghemoglobin can also be produced by genetically
engineering a bacterium or fungus to produce it. One illustrative
example is shown in FIG. 7. FIG. 7 shows stained SDS-PAGE gel
analysis of (A) soybean leghemoglobin expressed and purified using
recombinant protein technology and (B) soybean leghemoglobin
purified from soybean root nodules. (A) Recombinant Soybean
leghemoglobin A carrying His-tag and TEV protease His-tag removal
site was expressed in E. coli BL21 strain and purified using
His-tag affinity chromatography (Talon resin, CloneTech). The left
lane contains molecular weight standards, the right lane contains
purified recombinant soybean leghemoglobin A (arrow). Expected
molecular weight of the recombinant soybean leghemoglobin A is 17.1
kDa. (B) SDS-PAGE gel of purified Soybean leghemoglobin from root
nodules. The left lane contains molecular weight standards, the
right lane contains purified soybean leghemoglobin A (arrow). Mass
spectrometry analysis of purified material determined that all four
soybean leghemoglobin isoforms are present, and are full length
(data not shown). Expected molecular weights (MW) of soybean
leghemoglobin isoforms range from MW15.4 to 15.8 kDa.
[0382] Leghemoglobin purified from soybean and fava root nodules,
respectively was tasted by a panel of volunteers and in each case
described as tasting like blood.
[0383] Leghemoglobin can be isolated from the roots nodules of
legumes such as soy beans, fava beans, cow peas, lima beans,
garbanzo beans, peas, lupine, lotus japonicum or other legumes. The
root nodule (for example root nodules from a pea plant) is obtained
and homogenized in an aqueous solution, soluble proteins including
leghemoglobin are recovered after insoluble matter is removed by
precipitation or filtration. Leghemoglobin can be purified by
selective precipitation and/or chromatography and/or the use of
molecules with specific affinity for leghemoglobin. (FIG. 1,
showing 100 mls of solution of leghemoglobin isolated from 30 grams
of pea root nodules).
[0384] Heme proteins, for example leghemoglobin, can be combined
with other plant based meat replica components. In some embodiments
the heme proteins are captured in a gel which contains other
components, for example lipids and or proteins. In some aspects a
multiple gels are combined with non-gel based heme proteins. In
some embodiments the combination of the heme proteins and the other
compounds of the consumable are done to insure that the heme
proteins are able to diffuse through the consumable. In some
embodiments the consumable is ed in a heme-protein containing
solution, for instance a leghemoglobin solution. In some
embodiments the consumable is soaked in a heme protein containing
solution, for instance a leghemoglobin solution for 1, 5, 10, 15,
20 or 30 hours. In some embodiments the consumable is soaked in a
heme containing solution, for instance a leghemoglobin solution for
1, 5, 10, 15, 30, or 45 minutes.
[0385] FIG. 8 shows an example of 6 cubes of a commercial meat
analog (Quorn chicken analog), about 1 cm on a side, 4 of which
(Left and lower right) have been soaked in a solution of about 10
mg/ml soybean leghemoglobin in 20 mM Potassium phosphate pH 7.4 and
100 mM NaCl; the remaining two (Upper right) were soaked in the
same buffer without leghemoglobin. Note the deep pink color of the
leghemoglobin-infused cubes in contrast to the pale tan color of
the un-infused cubes.
[0386] FIG. 9 shows the 4 leghemoglobin-infused cubes of Quorn
chicken analog in the process of cooking in a pan at 350.degree. C.
The two lower cubes have been turned over to expose the grilled
surface, which has turned brown. Note in the upper two cubes that
the heated portion has turned grey-brown, while the cooler top
surface retains its pink color. In some embodiments the consumable
is injected with a heme containing solution, for instance a
leghemoglobin solution, until the consumable is the color of
uncooked meat.
[0387] Given the usefulness of heme proteins for coloring
consumables it will be useful to detect whether a product contains
a particular heme protein. Accordingly the present invention
includes in some embodiments methods to determine whether a product
contains a heme protein. Methods for detecting proteins are well
known in the art. For example an ELISA or proximity-ligation assay
or luninex assay or western blot analysis can be performed to
determine whether leghemoglobin is present in a food product such
as meat or a meat replica. In one embodiment the detection methods
are performed to determine whether meat has been altered with
leghemoglobin.
EXAMPLES
[0388] An exemplary muscle replica composition comprising one or
more isolated, purified plant proteins is described herein.
[0389] Protein Purification for Components of the Replica
[0390] Moong bean seeds, Green Pea dry seed were purchased as
milled flour and used for purification of respective seed storage
proteins. Rubsico was purified from fresh alfalfa plant. Protein
composition at individual fractionation steps was monitored by
SDS-PAGE and protein concentrations were measured by standard
UV-VIS and Pierce assay methods.
[0391] Moong bean 8S globulins: Moong bean flour was resuspended in
in 50 mM potassium phosphate buffer pH 7 and 0.5M NaCl at 1:4
(wt/v) ratio, and mixture was incubated for 1 hr. Unsoluble
material was separated by centrifugation and proteins in the
supernatant were fractionated by addition of ammonium sulfate in 2
steps: 50% (wt/v) followed by 90% (wt/v). Protein precipitated in
90% fraction contained the moong bean 8S globulins and was stored
at -20 C until further use.
[0392] Pea-albumins: Green pea dry seed flour was resuspended at
1:10 (wt/v) ratio in 50 mM sodium acetate buffer pH 5 and incubated
for 1 hr. Unsoluble material was separated by centrifugation and
proteins in the supernatant were fractionated by ammonium sulfate
precipitation in two steps: 50% (wt/v) followed by 90% (wt/v).
Ammonium sulfate solutions were stirred for 1 hour and ammonium
sulfate precipitated proteins removed by centrifugation. Proteins
of interest precipitated in 90% (wt/v) ammonium sulfate. Pellet was
stored at -20 C until further use.
[0393] Pea-globulins: Green pea dry seed flour was resuspended at
1:10 (wt/v) ratio in 20 mM potassium phosphate buffer pH 8, 0.4M
sodium chloride and stirred for 1 hr. After centrifugation, the
supernatant was subjected to ammonium sulfate fractionation. First,
supernatant was brought to 50% (wt/v) ammonium sulfate, and
precipitated proteins removed. Second, 50% (wt/v) ammonium sulfate
supernatant was brought to 80% (wt/v) ammonium sulfate saturation.
The 80% (wt/v) ammonium sulfate pelleted protein contained
globulins of interest. Pellet was stored at -20.degree. C. until
further use.
[0394] RuBisCO: RuBisCO was fractionated from alfalfa greens (or
other green plants eg soybean plants, spinach etc) by first
grinding leaves with 4 volumes of cold 50 mM KPhosphate buffer pH
7.4 buffer (with (in lab) or without (in field) 0.5M NaCl+2 mM
DTT+1 mM EDTA) in a blender. The resulting slurry was centrifuged
to remove debris, and the supernatant (crude lysate) was used in
further purification steps. Proteins in the crude lysate were
fractionated by addition of ammonium sulfate to 30% (wt/v)
saturation. The solution was stirred for 1 hr and then centrifuged.
The pellet from this step was discarded and additional ammonium
sulfate was added to the supernatant to 50% (wt/v) ammonium sulfate
saturation. The solution was centrifuged again after stirring for 1
hr. The pellet from this step contains RuBisCO, and was kept at -20
C until used.
[0395] Obtaining Plant Proteins.
[0396] Moong bean seed 8S protein was purified by ammonium sulfate
fractionation as described. Pellet was resuspended in 20 mM
potassium phosphate pH pH 7.4 and 0.5M sodium chloride and ammonium
sulfate removed by dialysis against the same buffer. Any
precipitate was removed by centrifugation at 16000 g, 10 min and
protein concentrated to desired concentration. Pea globulins
purified by ammonium sulfate fractionation as described. Protein
pellet was resuspended in 20 mM potassium phosphate pH pH 7.4 and
0.4M sodium chloride and ammonium sulfate removed by dialysis
against the same buffer. Any precipitate was removed by
centrifugation at 16000 g, 10 min and protein concentrated to
desired concentration. Pea albumin purified by ammonium sulfate
fractionation as described. Protein pellet was resuspended in 20 mM
potassium phosphate pH pH 7.4 and 0.1M sodium chloride and ammonium
sulfate removed by dialysis against the same buffer. Any
precipitate was removed by centrifugation at 16000 g, 10 min and
protein concentrated to desired concentration.
[0397] Constructing a Muscle Tissue Analog
[0398] Moong bean seed 8S protein was purified by ammonium sulfate
fractionation as described above, For preparation of gels, 200 g of
pellet was dissolved in 400 ml of dialysis buffer (20 mM potassium
phosphate, 400 mM NaCl, pH 7.3) and the resulting solution dialyzed
for 6 hours against 51 of dialysis buffer, replaced twice with
fresh buffer. Protein solution was centrifuged at 12,000 g for 15
min to remove debris. Protein was concentrated by dialyzing for 36
hours against 51 of 30% w/w solution of PEG 8000 (polyethylene
glycol, molecular weight 8000) in dialysis buffer. Final protein
concentration was 150 mg/ml.
[0399] Leghemoglobin was purified from soybean root nodules. Legume
root nodules were cleaned to remove soil and extraneous root
tissues prior to root nodule lysis in 20 mM potassium phosphate pH
7.4, 100 mM sodium chloride, 1 mm EDTA and 1 mM ascorbic acid. Root
nodules were lysed by grinding root-nodules using juicer blender.
Unsoluble material was separated by centrifugation. Root nodule
lysate was fractionated using ammonium sulfate in two steps, first
ammonium sulfate was added to 60% wt/v and solution incubated for 1
hr, 4.degree. C. Pellet was discarded and supernatant brought to
90% wt/v ammonium sulfate and incubated for 12 hr, 4.degree. C.
Leghemoglobin was collected as a precipitated pellet in 90%
ammonium sulfate fraction and resuspended in 20 mM potassium
phosphate, 1 mM EDTA, 100 mM sodium chloride. SDS-PAGE gel analysis
determined that protein solution contains 70% leghemoglobin and 30%
other root nodule proteins. Ammonium sulfate was removed using
size-exclusion chromatography in the same buffer. Leghemoglobin was
concentrated by dialyzing for 48 hr against 30% PEG 8000
(polyethylene glycol, molecular weight 8000) in 20 mM potassium
phosphate pH 7.3, 100 mM sodium chloride. Total protein
concentration was 57 mg/ml. UV-VIS spectra suggested that
leghemoglobin was in heme-iron oxidized state. Thus, leghemoglobin
was incubated with 5 mM sodium hydrosulfite for 5 min and sodium
hydrosulfite was removed using size-exclusion chromatography in 20
mM potassium phosphate, 100 mM sodium chloride buffer.
Leghemoglobin was further concentrated to 35.4 mg/ml. UV-VIS
spectra analysis confirmed that leghemoglobin is in heme-iron
reduced state.
[0400] Transglutaminase was obtained commercially from (Activa TI,
Ajimoto). Stock solution (20% wt/v) was made in 20 mM potassium
phosphate pH 7.3, 100 mM sodium chloride buffer.
[0401] To prepare "dark" muscle tissue analog (FIG. 10), 43 ml of
moong bean protein solution (150 mg/ml in dialysis buffer) were
mixed with 37 ml of leghemoglobin solution (46.5 mg/ml
leghemoglobin and 20 mg/ml of other soybean root nodule protein) in
20 mM potassium phosphate, 100 mM NaCl, pH 7.3). 20 ml of
transglutaminase solution (20% w/w) were added, solutions
thoroughly mixed, divided into two 50 ml Falcon tubes and incubated
overnight at room temperature. Final protein concentrations were 65
mg/ml for moong bean protein, 18 mg/ml of leghemoglobin, 91 mg/ml
total protein.
[0402] "White" muscle analog (FIG. 11) was prepared by mixing 43 ml
moong bean protein solution (150 mg/ml) with 45 ml of 11.7 mg/ml
solution of leghemoglobin and 0.8% (wt/v) of transglutaminase
solution. Final protein concentrations were 63 mg/ml for moong bean
protein, 5.2 mg/ml of leghemoglobin, 68 mg/ml total protein.
[0403] The "dark" muscle tissue analog formed an opaque gel of dark
chocolate color, smooth uniform texture, with glistening surface,
and a small amount (<1 ml) of dark red, venous blood colored
liquid on top. The gel was freely standing, elastic but fragile,
similar in appearance to thin Jell-O. The gel has a medium aroma
with notes of beans and blood clearly discernible. The flavor is
dominated by notes of beans and iron/blood, with weaker grassy and
medicinal/chemical flavors. The taste is salty, with a long
aftertaste of blood.
[0404] The "white" muscle tissue analog was very similar, but with
much lighter, cappuccino-like, color. It was also more fragile,
2-3-fold less strong against compression.
[0405] Fat Tissue Analog
[0406] Fat tissue analog using moong bean 8S globulin fraction was
prepared as follows: 15 ml of moong bean protein solution (150
mg/ml in dialysis buffer) were mixed with 15 ml of rice bran oil. 6
ml of transglutaminase solution (20% w/w) were added, solutions
thoroughly emulsified using a homogenizer (VWR) at speed #2.
Emulsion was aliquoted into 1.6 eppendorf tubes and incubated
overnight at room temperature. After that, tubes were heated at
95.degree. C. for 5 min in a heat block, and allowed to cool down
to room temperature on a bench. Final concentrations were 75 mg/ml
for moong bean protein, 50% w/w oil.
[0407] Fat tissue analog using pea globulin (100 mg/ml) was
prepared by the same method. Additionally, fat tissue analog was
prepared from pea globulin, and either rice bran or canola oil, in
bulk by the same method, but without aliquoting emulsions into
eppendorf tubes. Instead, emulsions in 50 ml Falcon tubes were
rotated overnight on a nutator, and were subsequently incubated at
90.degree. C. for 30 min.
[0408] Fat tissue analog based on moong beans (FIG. 12) and
prepared in eppendorf tubes formed an opaque gel of off-white
color, smooth uniform texture, with no visible discernible liquid
that was not incorporated into the gel. The gel was freely
standing, elastic and springy. The gel has a slight, pleasant aroma
and a mild and pleasant flavor. The taste is mildly salty.
[0409] Fat tissue analog based on pea globulin (FIG. 13) and
prepared in eppendorf tubes was very similar to moong bean-based
fat analog, except that it gave up a little of oil upon
compression. Fat tissue analog prepared in 50 ml Falcon tubes were
similar in appearance, texture and aromas, but substantially softer
(2-fold softer for canola oil, and 3-fold softer for rice bran oil,
according to compressibility measurements).
[0410] Connective Tissue Analog
[0411] Connective tissue analog prototypes were developed using
zein protein sourced from 100% yellow corn gluten meal, or from
commercial sources, such as Amazein (Prairie Gold, Bloomington,
Ill.). Zein proteins were solubilized in 70-90% ethanol with
desired ratios at 1:3 to 1:5 (solids:solution). By precipitating
zein proteins, for example by a change in pH, in a controlled
manner, large zein structures result with physicochemical
properties that can be manipulated as desired. For example, FIG. 14
shows connective-tissue analog strands that were created using a
1:3 ratio in 70% ethanol, loaded into a syringe with a 23 gauge
needle (ID 0.337 mm). The solution was slowly extruded from the
bottom of a 5 inch-high vessel into an excess of 5 M NaCl solution.
The ethanol-zein solution being less dense than the NaCl solution,
floated upward, drawing out a fibrous stand of solidifying zein.
The NaCl was constantly stirred as the strands began to develop to
assist in the strand lengthening. The strands bunch together and
become a hard, dense mass.
[0412] Ground Beef Replica Prototypes Made from Gels of Plant
Proteins and Plant Oils.
[0413] A ground beef prototype patty was made by combining 62%
(wt/wt) muscle analog (62% (wt/wt) "dark muscle analog" and 38%
(wt/wt) "white muscle analog"), 29% (wt/wt) fat tissue analog (from
pea globulin and canola oil), 5% (wt/wt) connective tissue analog
(FIG. 15 panel A). A ground beef prototype patty was made by
combining 62% muscle analog (62% "dark muscle analog" and 38%
"white muscle analog), 29% fat tissue analog (from moong bean seed
8S protein and rice bran oil), 5% connective tissue analog (FIG. 15
panel B). A ground beef prototype patty was made by combining 71%
(wt/wt) muscle tissue analog (composed of 60% "white" muscle
analog, 40% "dark" muscle analog), 23% fat tissue (from pea seed
globulin proteins and canola oil) (FIG. 15 panel C). A ground beef
prototype patty was made by combining 67% "White" muscle analog,
with 28% fat tissue analog (from pea globulins and rice bran oil),
(FIG. 15, panel D)
[0414] In a further test, the effect of cooking the ground beef
replica patties was evaluated by grilling on a 350.degree. F. pan.
A ground beef patty analog was made by combining 62% (wt/wt) muscle
tissue analog (62% (wt/wt) "dark muscle analog" and 38% (wt/wt)
"muscle analog"), 29% (wt/wt) fat tissue analog (from pea globulin
and canola oil), 5% (wt/wt) connective tissue analog (FIG. 16). The
panel on the left shows the patty before cooking and the panel on
the right shows the same patty after cooking for about 2 minutes.
Observers described the aroma of the cooking ground beef replica as
distinctly "beefy".
Sequence CWU 1
1
31144PRTGlycine max 1Met Val Ala Phe Thr Glu Lys Gln Asp Ala Leu
Val Ser Ser Ser Phe1 5 10 15 Glu Ala Phe Lys Ala Asn Ile Pro Gln
Tyr Ser Val Val Phe Tyr Thr 20 25 30 Ser Ile Leu Glu Lys Ala Pro
Ala Ala Lys Asp Leu Phe Ser Phe Leu 35 40 45 Ala Asn Gly Val Asp
Pro Thr Asn Pro Lys Leu Thr Gly His Ala Glu 50 55 60 Lys Leu Phe
Ala Leu Val Arg Asp Ser Ala Gly Gln Leu Lys Ala Ser65 70 75 80 Gly
Thr Val Val Ala Asp Ala Ala Leu Gly Ser Val His Ala Gln Lys 85 90
95 Ala Val Thr Asp Pro Gln Phe Val Val Val Lys Glu Ala Leu Leu Lys
100 105 110 Thr Ile Lys Ala Ala Val Gly Asp Lys Trp Ser Asp Glu Leu
Ser Arg 115 120 125 Ala Trp Glu Val Ala Tyr Asp Glu Leu Ala Ala Ala
Ile Lys Lys Ala 130 135 140 2133PRTMethylacidiphilum infernorum
2Met Ile Asp Gln Lys Glu Lys Glu Leu Ile Lys Glu Ser Trp Lys Arg1 5
10 15 Ile Glu Pro Asn Lys Asn Glu Ile Gly Leu Leu Phe Tyr Ala Asn
Leu 20 25 30 Phe Lys Glu Glu Pro Thr Val Ser Val Leu Phe Gln Asn
Pro Ile Ser 35 40 45 Ser Gln Ser Arg Lys Leu Met Gln Val Leu Gly
Ile Leu Val Gln Gly 50 55 60 Ile Asp Asn Leu Glu Gly Leu Ile Pro
Thr Leu Gln Asp Leu Gly Arg65 70 75 80 Arg His Lys Gln Tyr Gly Val
Val Asp Ser His Tyr Pro Leu Val Gly 85 90 95 Asp Cys Leu Leu Lys
Ser Ile Gln Glu Tyr Leu Gly Gln Gly Phe Thr 100 105 110 Glu Glu Ala
Lys Ala Ala Trp Thr Lys Val Tyr Gly Ile Ala Ala Gln 115 120 125 Val
Met Thr Ala Glu 130 3121PRTTetrahymena thermophile 3Met Arg Lys Gln
Pro Thr Val Phe Glu Lys Leu Gly Gly Gln Ala Ala1 5 10 15 Met His
Ala Ala Val Pro Leu Phe Tyr Lys Lys Val Leu Ala Asp Asp 20 25 30
Arg Val Lys His Tyr Phe Lys Asn Thr Asn Met Glu His Gln Ala Lys 35
40 45 Gln Gln Glu Asp Phe Leu Thr Met Leu Leu Gly Gly Pro Asn His
Tyr 50 55 60 Lys Gly Lys Asn Met Ala Glu Ala His Lys Gly Met Asn
Leu Gln Asn65 70 75 80 Ser His Phe Asp Ala Ile Ile Glu Asn Leu Ala
Ala Thr Leu Lys Glu 85 90 95 Leu Gly Val Ser Asp Gln Ile Ile Gly
Glu Ala Ala Lys Val Ile Glu 100 105 110 His Thr Arg Lys Asp Cys Leu
Gly Lys 115 120
* * * * *