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WO2003007728A2 - Denree alimentaire proteique vegetarienne - Google Patents

Denree alimentaire proteique vegetarienne Download PDF

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Publication number
WO2003007728A2
WO2003007728A2 PCT/EP2002/008071 EP0208071W WO03007728A2 WO 2003007728 A2 WO2003007728 A2 WO 2003007728A2 EP 0208071 W EP0208071 W EP 0208071W WO 03007728 A2 WO03007728 A2 WO 03007728A2
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WO
WIPO (PCT)
Prior art keywords
protein
enzyme
edible foodstuff
cross
produced
Prior art date
Application number
PCT/EP2002/008071
Other languages
English (en)
Other versions
WO2003007728A3 (fr
Inventor
Robert Franciscus Beudeker
Albertus Alard Van Dijk
Original Assignee
Dsm Ip Assets B.V.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Dsm Ip Assets B.V. filed Critical Dsm Ip Assets B.V.
Priority to AU2002331272A priority Critical patent/AU2002331272A1/en
Publication of WO2003007728A2 publication Critical patent/WO2003007728A2/fr
Publication of WO2003007728A3 publication Critical patent/WO2003007728A3/fr

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Classifications

    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
    • A23J3/00—Working-up of proteins for foodstuffs
    • A23J3/20—Proteins from microorganisms or unicellular algae
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/17—Amino acids, peptides or proteins
    • A23L33/195—Proteins from microorganisms

Definitions

  • the present invention relates to edible foodstuffs especially vegetarian protein foodstuffs comprising fungal derived protein which has been enzymaticaily cross linked and in particular to novel processes for the production of edible foodstuffs comprising fungal derived protein which has been enzymaticaily crossed linked.
  • Such foodstuffs have acceptable taste, texture and are devoid of any animal products and so are suitable for vegetarian and/or vegan consumption.
  • fungi e.g. mushrooms
  • proteinaceous foods containing fungi are known.
  • One example is the traditional Indonesian fermented food, tempeh. This is usually prepared by the fermentation of Rhizopus fungi with soy beans (and parts thereof) acting as a moist substrate. The beans (or other vegetable substrates) are inoculated with the fungus and fermentation allowed for 24 to 36 hours. The beans become bound by the fungal mycelium protein to give a firm product which can then be sliced before eating; no additional processing is usually performed before consumption.
  • the disadvantage of tempeh over meat is its lack of taste, flavour and fibrous and juicy texture which is associated with meat.
  • Soy-based products in particular extruded soy, are marketed, but they do not have a particularly meat-like taste or texture (indeed both soy and gluten can have an "off" or astringent taste).
  • Egg albumin is derived from eggs which are produced by animals making this food unacceptable to vegans because of ethical objections. Eggs may also contain contaminants such as dioxin and human pathogenic bacteria such as Salmonella.
  • the present invention provides an edible foodstuff comprising protein based on fermentatively produced fungi wherein the fungal protein has been enzymaticaily crossed linked.
  • Such foodstuffs may or may not comprise a protein cross-linking enzyme.
  • the present invention also provides a process of production of an edible foodstuff, which comprises the addition of a protein cross-linking enzyme to an industrially produced fungal biomass containing protein.
  • the present invention relates to the use of protein cross-linking enzymes, such as transglutaminase, to improve the texture of edible proteinaceous foodstuffs based on fungal cells. It is now possible to obtain a desirable texture without the addition of proteins of animal origin, for example the addition egg albumin to the fugal mycelium, during food preparation.
  • protein cross-linking enzymes such as transglutaminase
  • vegetarian protein foodstuffs based on fungal cells can be prepared which have an improved texture by the use of cross- linking enzymes derived from, produced by or present in a micro-organism.
  • the cross-linking enzyme e.g. transglutaminase
  • the cross-linking enzyme is preferably recombinantly produced, such as by heterologous expression of an encoding gene or cDNA in a suitable host organism, or, alternatively, by homologous (e.g. over expression) of a suitable endogenous gene.
  • the enzyme may be produced by a (wild-type) strain of e.g. a Streptoverticillium species.
  • Illustrative strains include Streptoverticillium griseocarneum IFO 1 2776, Streptoverticillium cinnamoneum sub sp. Cinnamomeum IFO 1 2852, Streptoverticillium mobaraense IFO 1 381 9 and other species (cf.
  • the enzyme may be produced (e.g. recombinantly by expression of a heterologous gene) by a micro-organism such as a bacteria, yeast or fungus (e.g. filamentous fungi) .
  • a micro-organism such as a bacteria, yeast or fungus (e.g. filamentous fungi) .
  • the organism is of the genus Streptomyces, Bacillus, Escherichia, Saccharomyces, Kluyvermyces, Hansenula, Pichia, Yarrowia, Candida, Aspergillus, Trichoderma, Penicillum, Mucor, Fusarium or Humicola.
  • Typical preferred (production) organims are E. coli, Streptomyces e.g.
  • Streptomyces lividans Bacillus e.g. Bacillus licheniformis, Saccharomyces e.g. Saccharomyces cerevisae, Kluyvermyces lactis and Aspergillus e.g. Aspergillus niger.
  • the enzyme can be produced by fermentation (of the organism) and then additional processing to recover the enzyme.
  • protein cross-linking enzymes which can be used according to the invention comprise:
  • Transglutaminase (EC 2.3.3,13) ; protein-glutamine gamma- glutamyltransferase) is an enzyme capable of catalyzing acyl-transfer reactions introducing covalent cross-links between proteins as well as peptides and various amino acids. Large scale production of transglutaminase is possible by cloning a
  • Protein disulfide isomerase (EC 5.3.4.1 ), for example, catalyzes the reduction of disulphide groups in protein to reactive sulphydryl residues. Oxidation of these sulphydryl groups results in the formation of covalent cross-links between two polypeptide chains or between two residues in the same chain.
  • Sulphydryl oxidase (EC 1 .8.3.2) or thiol oxidase is capable of catalyzing the oxidation of sulphydryl groups in proteins to disulfide bonds. This enzyme is found in both animals and microorganisms.
  • Polyphenol oxidase (EC 1 .14.1 8.; previously classified as EC 1 .10.3.1 ) is also known as catechol oxidase, tyrosine oxidase, tyrosinase, phenolase or phenol oxidase.
  • This enzyme is widespread in nature and is found in many different types of animals such as e.g. mammals, insects, fish, moluscs, nematodes, plants and microorganisms.
  • the enzyme catalyzes the oxidation of phenols to ortho-diphenols which are subsequently oxidised to ortho-diquinone by the same enzyme.
  • the ortho- diquinones react with sulphydryl groups or with amine groups within the protein to form protein cross-links.
  • Lysyl oxidase (EC 1 .4.3.1 3) of lysyl-protein-6-oxidase is an important enzyme in the formation of protein cross-linking in collagen and elastin. Lysyl oxidase catalyzes the oxidative deamination of lysine to alpha-amino adipic-delta- semialdehyde or its hydroxide. These reactive molecules react with other amino acids to form cross links.
  • Peroxidase (EC 1 .1 1 .1 .7.) is capable of catalyzing a large number of reactions in proteins in the absence or presence of hydrogen peroxide. Hydroxylation and peroxidation activities of peroxidase cause protein cross-linking. However cross- linking enzymes other than peroxidase(s) can be used depending on the circumstances.
  • Lipoxygenase (EC 1 .1 3.1 1 .1 2) also lipoxydase or linolate:oxygen oxidoreductase catalyses the oxidation of, unsaturated fatty acids to fatty acid peroxo radicals. These reactive molecules cause protein cross-linking.
  • oxidases produce hydrogen peroxide and cause protein cross- linking in an indirect way.
  • Glucose oxidase (EC 1 .1 .3.4) for example, catalyses the oxidation of glucose to gluconolactone and hydrogen peroxide which in its turn oxidizes proteins.
  • Alcoholoxidases as e.g. methanoloxidase may result in protein cross-linking through the formation of aldehydes (formaldehyde in the case of methanol oxidase) .
  • a polymeric or oligomeric carbohydrate with oxidizable groups for example ferulic acid.
  • plant or micriobal polymeric or oligomeric carbohydrates are used.
  • Xylans present in, for example, wheat often contain such oxidizable groups. Addition of an oxidase to a medium containing such carbohydrates will result in the cross-linking of proteins with this carbohydrate.
  • the fungal cells, fungal biomass or fungal protein is in general produced on industrial scale for example in a fermentor.
  • the fungi can be of the family Choanephoraceae, such as of the genus
  • Blakes/ea or Gilbertella for example of the species Blakeslea trispora or Gilbertella persicaria.
  • the other three families included within the order Mucorales are Cunninghame/laceae, Mortiere llaceae (such as fungi of the genus Mortierella, and in particular the species Mortierella alpina) and, especially, Mucoraceae. Suitable fungi are usually edible (and digestible) by humans or animals.
  • Preferred fungi are saprophytic (that is to say, simple fungi) rather than parasitic (which are more complex).
  • the "simple" fungi are usually preferred because they are better adapted towards hyphal growth, whereas the parasitic organims concentrate on taking nutrients from their "host” organism.
  • the fungal cells are preferably of the genus Rhizopus, Rhizomucor, Mucor or
  • Rhizopus Suitable fungal of the genus Rhizopus, Mucor or Rhizomucor include Rhizopus stolonifer, Rhizopus miehei, Rhizopus pusillus, Rhizopus oligosporus and in particular, Rhizopus oryzae; Mucor hiema/is and Mucor rouxii; and Rhizomucor meihei.
  • Other preferred strains include those of the genus Absidia or Phycomyces, such as Absidia pseudocylindrospora or Phyzomyces blakesleeanus.
  • the addition of the protein cross-linking enzyme preferentially takes place during mixing of the fungal biomass and flavours but before forming and steaming.
  • the steam-cooking after forming and addition of the cross-linking enzyme results in rapid inactivation of the enzyme activity.
  • a complete inactivation of the enzyme is preferred in order to prevent further enzyme activity during consumption of the resulting product.
  • Streptomyces lividans transformants expressing the Streptoverticillium transglutaminase gene were grown in a complex medium as described in this patent.
  • the final fermentation broth had transglutaminase activities ranging from 1 -1 0 U/ml
  • the microorganism was separated from the broth by means of centrifugation for 10 min. at 1 2,000 g at 4°C.
  • the liquid containing the enzyme was concentrated by means of ultrafiltration (cut off was 1 ,000 Da) to a final enzyme concentration of
  • Enzyme analysis was then carried - out as described in J.Biol.Chem. 241 : 551 8 ( 1 966) using benzyloxycarbonyl-L-glutamylglycine and hydroxylamine as substrates.
  • One unit of enzyme activity is defined as the amount of enzyme catalyzing the formation of one 1 micromole of hydroxamic acid per minute at 37°C.
  • Rhizopus oryzae (own isolate; other strains of this species are available from the Centraal Bureau voor Schimmelcultures, Delft, the Netherlands) was grown on labscale as described in WO 00/1 5045.
  • a spore suspension of the strain was prepared by growing the fungus for several days on a malt agar surface, rinsing the spores from the surface and storing them in a freezer. With this spore suspension an inoculum culture was started, using a soy bean meal based medium to promote hyphal growth (soy flour 1 5g/kg, yeast extract 5g/kg, K 2 HPO 4 1 g/kg and glucose. H 2 0 20g/kg) . The medium was sterilized for 45 min. at 1 20°C in Erlenmeyer flasks at pH 6. The flasks were incubated between 25 and 35°C for 2 to 4 days on an orbital shaker (with a 2.5 cm stroke at 250 rpm). As soon as full growth had been reached the culture was transferred to a lab fermentor containing a batch medium that was was prepared using the following components with indicated final concentrations:
  • Yeast extract (1 g/kg), glucose (20g/kg), ammonium sulphate (6 g/kg), magnesium sulphate.7H 2 0 (2 g/kg), calcium chloride (0.5 g/kg), potassium monophosphate (3 g/kg), zinc sulphate.7H 2 0 (0.01 4g/kg), iron sulphate (0.1 5 g/kg), manganese sulphate.1 H 2 0 (0.0288g/kg), copper sulphate.5H 2 0 (0.0024g/kg), cobalt sulphate.7H 2 0 (0.0038g/kg), thiamine.HCI (0.004 g/kg) and nicotinic acid (0.002 g/kg).
  • a carbohydrate feed was supplied which consisted of glucose at a concentration of ca. 500 g/kg.
  • the preparation was as described for the glucose solution of the batch medium.
  • the fermentor was equipped with temperature, pH and foam controls. To adjust the pH, solution of ammonia and sulphuric acid were used. Dissolved oxygen concentration and the composition of the liberated gas were measured.
  • the culture was aerated using ca. 1 volume of air per volume of broth per minute. Mixing was intensive using Rushton turbines and baffles.
  • the glucose feed was applied at a rate between 1 and 5 g of glucose/kg borth/hour and started when the glucose concentration in the broth had decreased., to a concentration below 5 g/kg.
  • the temperature during fermentation was 30-35°C, the starting pH 4 and fermentation lasted for about 80 hours.
  • the biomass was separated from the broth by means of centrifugation (5 min. at 5000 rpm) and the biomass was then subjected to filtration on a lab filter press to remove excess water.
  • the cake was then frozen at -20°C for further treatment.
  • Comparative Examples 3 and 5 + Example 4 The filter cake from Comparative Example 2 was milled and crumbled by a high shear mixer for 5 minutes. The crumbled cake was subjected to the following three treatments:
  • Enzyme dosages vary between 1 0,000 and 1 0 U/kg of protein treated.
  • Patties were then made according to the following protocol: 100 g of the homogenized mixture was mixed with 4.6 g soy oil in a labscale food processor (Braun Combi type 700) . The resulting dough was placed in moulds and heated to 85°C by steaming during 5 minutes. After chilling to 4-7°C the patties were frozen to -20°C.

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  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Polymers & Plastics (AREA)
  • Health & Medical Sciences (AREA)
  • Nutrition Science (AREA)
  • Food Science & Technology (AREA)
  • Microbiology (AREA)
  • Mycology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biotechnology (AREA)
  • Cell Biology (AREA)
  • Biochemistry (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Enzymes And Modification Thereof (AREA)

Abstract

L'invention concerne une denrée alimentaire comestible comprenant une protéine à base de champignons fermentés, la protéine fongique étant réticulée de manière enzymatique.
PCT/EP2002/008071 2001-07-18 2002-07-18 Denree alimentaire proteique vegetarienne WO2003007728A2 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU2002331272A AU2002331272A1 (en) 2001-07-18 2002-07-18 Vegetarian protein foodstuff

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
EP01000305.1 2001-07-18
EP01000305 2001-07-18

Publications (2)

Publication Number Publication Date
WO2003007728A2 true WO2003007728A2 (fr) 2003-01-30
WO2003007728A3 WO2003007728A3 (fr) 2003-12-18

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AU (1) AU2002331272A1 (fr)
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Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004026039A1 (fr) * 2002-09-17 2004-04-01 Marlow Foods Limited Composition comportant un compose proteique, sa preparation et son utilisation
WO2006084953A1 (fr) * 2005-02-10 2006-08-17 Valtion Teknillinen Tutkimuskeskus Nouvelles enzymes microbiennes et leur utilisation
US8048652B2 (en) 2005-05-12 2011-11-01 Martek Biosciences Corporation Biomass hydrolysate and uses and production thereof
GB2516491A (en) * 2013-07-24 2015-01-28 Marlow Foods Ltd Edible Fungi
WO2016120594A1 (fr) * 2015-01-27 2016-08-04 Marlow Foods Limited Champignons comestibles
CN112471322A (zh) * 2011-07-12 2021-03-12 非凡食品有限公司 用于消费品的方法和组合物
US20220007777A1 (en) * 2018-11-14 2022-01-13 Bolt Threads, Inc. Methods of generating mycelium materials with improved properties
SE2151533A1 (en) * 2021-12-15 2023-06-16 Mycorena Ab Fungal biomass food product
US11819041B2 (en) 2014-03-31 2023-11-21 Impossible Foods Inc. Ground meat replicas
WO2023227743A1 (fr) 2022-05-25 2023-11-30 Bumble Be Gmbh Substitut d'aliment mycélien et son procédé de production
CN118389299A (zh) * 2024-05-24 2024-07-26 上海雅酶生物医药科技有限公司 一种真菌蛋白肉生产用毛霉菌种的制备方法及真菌蛋白肉
JP7667871B2 (ja) 2021-04-27 2025-04-23 ソーラー フーズ ユルキネン オサケユイチア 肉類似食品及びその製造方法

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB8308162D0 (en) * 1983-03-24 1983-05-05 Ranks Hovis Mcdougall Plc Edible protein containing substances
AU4262399A (en) * 1998-05-14 1999-11-29 Dsm N.V. Use of protein cross-linking enzymes in ruminant feed
EP0986960A1 (fr) * 1998-09-15 2000-03-22 Dsm N.V. Champignons mucorales pour l'utilisation dans la préparation de produits texturées pour des aliments

Cited By (20)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004026039A1 (fr) * 2002-09-17 2004-04-01 Marlow Foods Limited Composition comportant un compose proteique, sa preparation et son utilisation
GB2409796A (en) * 2002-09-17 2005-07-13 Marlow Foods Ltd Composition comprising proteinaceous material, its preparation and use
GB2409796B (en) * 2002-09-17 2006-06-07 Marlow Foods Ltd Composition comprising proteinaceous material, its preparation and use
WO2006084953A1 (fr) * 2005-02-10 2006-08-17 Valtion Teknillinen Tutkimuskeskus Nouvelles enzymes microbiennes et leur utilisation
AU2006212149B2 (en) * 2005-02-10 2010-03-04 Valtion Teknillinen Tutkimuskeskus Novel microbial enzymes and their use
US7910344B2 (en) 2005-02-10 2011-03-22 Valtion Teknillinen Tutkimuskeskus Proteins having tyrosinase activity
US8048652B2 (en) 2005-05-12 2011-11-01 Martek Biosciences Corporation Biomass hydrolysate and uses and production thereof
CN112471322A (zh) * 2011-07-12 2021-03-12 非凡食品有限公司 用于消费品的方法和组合物
GB2516491A (en) * 2013-07-24 2015-01-28 Marlow Foods Ltd Edible Fungi
GB2518726A (en) * 2013-07-24 2015-04-01 Marlow Foods Ltd Edible fungi
GB2516491B (en) * 2013-07-24 2015-06-17 Marlow Foods Ltd Edible Fungi
GB2518726B (en) * 2013-07-24 2020-05-27 Marlow Foods Ltd Edible fungi
US11819041B2 (en) 2014-03-31 2023-11-21 Impossible Foods Inc. Ground meat replicas
WO2016120594A1 (fr) * 2015-01-27 2016-08-04 Marlow Foods Limited Champignons comestibles
JP2018506300A (ja) * 2015-01-27 2018-03-08 マーロウ フーズ リミテッドＭａｒｌｏｗ Ｆｏｏｄｓ Ｌｉｍｉｔｅｄ 食用菌
US20220007777A1 (en) * 2018-11-14 2022-01-13 Bolt Threads, Inc. Methods of generating mycelium materials with improved properties
JP7667871B2 (ja) 2021-04-27 2025-04-23 ソーラー フーズ ユルキネン オサケユイチア 肉類似食品及びその製造方法
SE2151533A1 (en) * 2021-12-15 2023-06-16 Mycorena Ab Fungal biomass food product
WO2023227743A1 (fr) 2022-05-25 2023-11-30 Bumble Be Gmbh Substitut d'aliment mycélien et son procédé de production
CN118389299A (zh) * 2024-05-24 2024-07-26 上海雅酶生物医药科技有限公司 一种真菌蛋白肉生产用毛霉菌种的制备方法及真菌蛋白肉

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Publication number Publication date
AU2002331272A1 (en) 2003-03-03
WO2003007728A3 (fr) 2003-12-18

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