WO2014102757A1 - Procédé d'obtention d'extraits peptidiques bioactifs et polysaccharidiques à partir d'un excédent de levure de bière et utilisations correspondantes - Google Patents

Procédé d'obtention d'extraits peptidiques bioactifs et polysaccharidiques à partir d'un excédent de levure de bière et utilisations correspondantes Download PDF

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Publication number
WO2014102757A1
WO2014102757A1 PCT/IB2013/061445 IB2013061445W WO2014102757A1 WO 2014102757 A1 WO2014102757 A1 WO 2014102757A1 IB 2013061445 W IB2013061445 W IB 2013061445W WO 2014102757 A1 WO2014102757 A1 WO 2014102757A1
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WIPO (PCT)
Prior art keywords
fractions
peptide
extracts
extraction
polysaccharide
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Application number
PCT/IB2013/061445
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English (en)
Portuguese (pt)
Inventor
Maria Manuela Estevez Pintado
Original Assignee
Escola Superior De Biotecnologia Da Universidade Católica Portuguesa
Centro De Farmacologia E Biopatologia Química Da Universidade Do Porto
Queijo Saloio – Indústria De Lacticínios, S.A.
Unicer - Bebidas, S.A
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Application filed by Escola Superior De Biotecnologia Da Universidade Católica Portuguesa, Centro De Farmacologia E Biopatologia Química Da Universidade Do Porto, Queijo Saloio – Indústria De Lacticínios, S.A., Unicer - Bebidas, S.A filed Critical Escola Superior De Biotecnologia Da Universidade Católica Portuguesa
Publication of WO2014102757A1 publication Critical patent/WO2014102757A1/fr

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/06—Fungi, e.g. yeasts
    • A61K36/062—Ascomycota
    • A61K36/064—Saccharomycetales, e.g. baker's yeast
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/06—Lysis of microorganisms
    • C12N1/063—Lysis of microorganisms of yeast
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14—Fungi; Culture media therefor
    • C12N1/16—Yeasts; Culture media therefor
    • C12N1/18—Baker's yeast; Brewer's yeast
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)

Definitions

  • the present invention relates to a process for producing peptide and polysaccharide concentrates derived from a brewer's yeast autolysate by enzymatic hydrolysis and filtration techniques.
  • the resulting concentrates of the process according to the present invention comprise in their constitution peptides, amino acids, polysaccharides with various biological activities, such as antihypertensive, antiinflammatory, antiulcerative, antioxidant, prebiotic, antimicrobial, obesity and which can be applied in the food (human and animal) and pharmaceutical industries.
  • Saccharomyces yeast is widely used in human culture, and in particular in the production of alcoholic beverages. They are a byproduct of the brewing industry after being used in the manufacture of the beverage for the transformation of sugar into alcohol and carbon dioxide. This by-product represents about 2 to 3% of total production. With a worldwide production of approximately 1575 million hectoliters of beer per year, 31.5 - 47 million hectoliters of yeast waste are accumulated (Jaehrig, et al., 2008). Much of this by-product is sold at a low price for incorporation into animal feed, or is disposed of as production waste, thus causing serious environmental problems. However, certain cellular components, for example, the cell wall structure - ⁇ -glucan polymer - have several biological activities and therefore represent a potential physiological value.
  • ⁇ -glucan polymers are believed to have several immunomodulatory properties. In vitro and in vivo studies reveal that this stimulatory activity depends on structure, molecular weight and number of bonds (Mantovani, et al., 2008), thus exerting a beneficial effect against a wide range of human pathogens, including , bacteria, viruses, fungi and parasitic infections (Shim, et al., 2005). These compounds also exhibit hypocholesterolemic, antioxidant and anticoagulant properties. Recently, studies have shown anticytotoxic, antimutagenic and antitumor activities, thus making them promising pharmacological candidates (Mantovani, et al., 2008).
  • peptides can be obtained by yeast extract, by autolysis, plasmolysis and hydrolysis methods, but the most common practice is autolysis (Kim et al., 1999).
  • hydrolytic enzymes mainly proteases and nucleases, break down macromolecules (proteins and nucleic acids) and produce soluble products such as peptides, amino acids (mainly glutamate), nucleotides and amino acid derivatives (Tanguler and Erten, 2007).
  • the present invention describes a process for obtaining peptide and / or polysaccharide extracts which comprises the following steps: a) beer yeast autolysis; preferably at 60-80 ° C for 3-4 hours while stirring; even more preferably at 70 ° C for 3 hours while stirring; b) ultrafiltration of the membrane autolysate, preferably with 10000 - 20000 Da pore membrane; still more than 3000 Da; c) hydrolysis of the obtained fractions - filtered and retained - by the action of at least one Cynara cardunculus flower extract enzyme, preferably for 3 hours; even more preferably the concentration of the C flower extract.
  • Candunculus range from 1% -5% (v / v), preferably 3% (v / v); d) nanofiltration of membrane hydrolyzed fractions; e) concentration of each fraction by reverse osmosis, preferably with membrane with pores of 1000 - 3000 Da.
  • the extraction process described further comprises a process of extracting betaglucans / polysaccharides from said ultrafiltration retentate and / or the initial fraction of autolysate of yeast be subjected additionally.
  • the beta-glucan / polysaccharide extraction process may comprise the following steps: a) centrifuging the fractions, preferably at 8000 rpm 30 min; b) extraction with sodium hydroxide, preferably at 80 ° C for 2 hours; ; c) washing the fractions, preferably with water by centrifugation 4000 G, for 10 min; d) acid extraction with acetic acid, preferably 0.5N, 75 ° C, for 1 hour; e) washing the fractions, preferably with water by centrifugation 4000 G, for 10 min; f) drying of the fractions.
  • Another aspect of the present invention is the peptide / polysaccharide extracts obtainable by the extraction processes described above, their use in the food industry and medicine. Namely, in the treatment of hypertensive diseases, infectious diseases, gastric diseases or neoplasms.
  • Another aspect of the present invention are compositions which have previously described peptide / polysaccharide extracts obtainable by the processes for obtaining peptide and polysaccharide extracts described above, namely pharmaceutical and / or cosmetic compositions.
  • Another aspect of the present invention are foods or food supplements or food additives which comprise the peptide / polysaccharide extracts described above and obtainable by the processes of obtaining peptide and polysaccharide extracts described above.
  • the present invention relates to a process for obtaining peptide and polysaccharide extracts by autolysis, ultrafiltration, and enzymatic hydrolysis present in an aqueous brewer's yeast extract of Cynara cardunculus.
  • the concentration of enzyme extract should be from 1 to 5% (v / v) preferably
  • the procurement process comprises the following steps:
  • the present invention further relates to the biological activity of these extracts such as inhibition of angiotensin converting enzyme, antioxidant, antiulcerative, prebiotic activity, decreased body weight gain and antimicrobial activity.
  • Said extracts may be used: in the preparation of extracts with antihypertensive activity, or preparations with pre-biotic antioxidant activity; antimicrobial
  • the peptide concentrate - fraction D shows in vitro antimicrobial activity for Escherichia coli, Staphylococcus aureus (MRSA) methicillin resistant Salmonella enteritidis (ATCC 3076) and Listeria innocua (11288 NCTC).
  • Figure 1 Schematic representation of the various steps of the process of obtaining bioactive and polysaccharide peptide extracts through the use of beer yeast.
  • the present invention relates to a process of preparing bioactive peptides, peptide extracts and polysaccharides from beer yeast autolysis, subsequent membrane filtration and hydrolysis of fractions by hydrolytic action of cardosines and similar enzymes. Also the development and optimization of a filtration process, which allows obtaining protein concentrates, peptides and polysaccharides with biological activities.
  • One aspect of the present invention is the use of these extracts in food and feed, and pharmaceuticals that have health benefits, particularly in the treatment and / or prevention of pathologies.
  • the excess brewer's yeast is subjected to a temperature range of 60 to 80 ° C, preferably 70 ° C in a stirred tank for 3-4 hours, preferably 3 hours.
  • Nano-Retained Ultrafiltration Retained (NRet-Ret) - A Nano-Retained Ultrafiltration Retained (NFil-Ret) -B Nano-Retained Ultrafiltration (NRet-Fil) - C Nano-filtered Ultrafiltration ( NFil-Fil) - D
  • Acid extraction with acetic acid preferably 0.5 N 75 ° C 1 hour;
  • the ACE inhibitor potential of biopeptide-rich extracts was performed using the modified Sentandreu and Toldrá (2006) method, which is based on the hydrolysis of the fluorescent substrate o-aminobenzoyl glycyl-p-nitrophenylalanylproline by ACE action. Such inhibition was evaluated for all hydrolysis extracts.
  • the compound used as an ACE substrate was Abz-Gly-Phe (NO2) -Pro (o-aminobenzoylglycyl-p-nitrophenylalanylproline), which was dissolved in 150 mM Tris-HCl buffer with pH 8.3. 1125 mM sodium to give a final concentration of 0.45 mM substrate at pH 8.3.
  • 160 ⁇ of 40 ⁇ substrate was mixed from each of the samples for which ACE inhibitory activity was to be determined, and 2 mU of the enzyme ACE dissolved in glycerol was added to the assay.
  • ACE inhibitory activity was calculated as the amount of soluble protein needed to inhibit 50% enzyme (IC 50 ) (in ⁇ g protein / mL). To be accomplished For this calculation, a nonlinear adjustment of the data was made using the PRISM v. 4.02 for Windows (GraphPad Software, Inc., San Diego, CA, USA). The activity of each sample was determined in triplicate. To calculate the inhibitory activity of each sample, the following formula (3) was used:
  • the blank received the same treatment as the samples, but instead of adding the enzyme, water was added;
  • the positive control was also subjected to the same treatment by placing 40 ⁇ of water instead of the sample.
  • mice Male SHR rats (spontaneously hypertensive) were used, young adults (10 weeks old, with installed hypertension).
  • the extracts tested were administered to the animals acutely by gavage (metallic, orogastric tube) at a dose of 300 mg / kg body weight, in a single dose.
  • Blood pressure was monitored by telemetry, with records of systolic and diastolic blood pressure values at the end of 2h, 4h, 8h and 24h.
  • blood was collected for test of tolerance to glucose and insulin (4 th and 10 th treatment week) monitored blood pressure by the tail cuff (2a, 8 th and 11 th treatment week) and collected adipose tissue, pancreas, liver, urine and feces (at the end of treatment).
  • Oxygen radical absorption capacity (ORAC - Fluorescein) was determined according to the method developed by Ou et al. (2001), adapted by Dávalos et al. (2004), using a microplate fluorescence reader, with some modifications. This method is based on the oxidation of fluorescein by peroxyl radicals produced in situ by thermal decomposition of 2,2'-azo-bis- (2-methylpropionamidine) dihydrochloride (AAPH). Fluorescein oxidation causes a decrease in fluorescence; However, this process can be avoided or delayed in the presence of antioxidant substances.
  • the fluorescein solution was prepared daily at a concentration of 116.66 nM from a fluorescein 1166.1 ⁇ stock solution in 75 mM phosphate buffer (pH 7.4).
  • As reference antioxidant 6-hydroxy-2,5,7,7-tetramethylchroman-2-carboxylic acid (Trolox) was used.
  • AUC 1+ fi / fo where fo is the initial fluorescence measured at time 0 min and fi is the fluorescence measured in a cycle i.
  • the sample AUC was calculated according to the formula:
  • AUC Antioxidant AUC - AUC Control
  • AUC was plotted against oxidant amount and linear regression was calculated.
  • the final value of 0RAC-F1 was expressed as the ratio between the slope of the sample curve and that of Trolox ( ⁇ Trolox equivalents / mg protein / peptide).
  • V79 fibroblast
  • 3T3 fibroblast
  • the 50% effective dose corresponds to the dose required to produce 50% inhibition in the observed lesions compared to the negative control.

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  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
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Abstract

La présente demande concerne un procédé d'obtention d'extraits peptidiques et polysaccharidiques d'un autolysat de levure de bière, par hydrolyse enzymatique et techniques de filtration. Elle concerne également les extraits peptidiques et polysaccharidiques obtenus au moyen dudit procédé, ainsi que leur utilisation à des fins thérapeutiques en médecine ou comme médicament dans le traitement de maladies hypertensives, de maladies infectieuses, de maladies gastriques ou de néoplasies. La présente demande concerne en outre des compositions, des aliments, des compléments et des additifs alimentaires comprenant lesdits extraits peptidiques et/ou polysaccharidiques.
PCT/IB2013/061445 2012-12-31 2013-12-31 Procédé d'obtention d'extraits peptidiques bioactifs et polysaccharidiques à partir d'un excédent de levure de bière et utilisations correspondantes WO2014102757A1 (fr)

Applications Claiming Priority (2)

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PT10671012 2012-12-31
PT106710 2012-12-31

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WO2014102757A1 true WO2014102757A1 (fr) 2014-07-03

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023119195A3 (fr) * 2021-12-21 2023-08-03 Universidade Católica Portuguesa - Ucp ÉMULSION DE β-GLUCANE DE LEVURE, SES MÉTHODES ET UTILISATIONS

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009007424A1 (fr) * 2007-07-10 2009-01-15 Dsm Ip Assets B.V. Autolysats de levure

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009007424A1 (fr) * 2007-07-10 2009-01-15 Dsm Ip Assets B.V. Autolysats de levure

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
ROLANDO CAMPOS ET AL: "Chemical Characterization of Proteases Extracted from Wild Thistle (Cynara cardunculus)", FOOD CHEMISTRY, vol. 35, 1 January 1990 (1990-01-01), pages 89 - 97, XP055113079 *
VESNA ZECHNER-KRPAN ET AL: "Characterization of b-Glucans Isolated from Brewer's Yeast and Dried by Different Methods", FOOD TECHNOL. BIOTECHNOL, vol. 48, no. 2, 1 January 2010 (2010-01-01), pages 189 - 197, XP055113075 *
ZÉLIA VELEZ ET AL: "Biological Characterization of Cynara cardunculus L. Methanolic Extracts: Antioxidant, Anti-proliferative, Anti-migratory and Anti-angiogenic Activities", AGRICULTURE, vol. 2, no. 4, 19 December 2012 (2012-12-19), pages 472 - 492, XP055113069, DOI: 10.3390/agriculture2040472 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023119195A3 (fr) * 2021-12-21 2023-08-03 Universidade Católica Portuguesa - Ucp ÉMULSION DE β-GLUCANE DE LEVURE, SES MÉTHODES ET UTILISATIONS

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