WO2014163482A1 - Gélose dextrosée à la protéine de palme (ppda) pour détecter et énumérer les champignons et la levure - Google Patents

Gélose dextrosée à la protéine de palme (ppda) pour détecter et énumérer les champignons et la levure Download PDF

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Publication number
WO2014163482A1
WO2014163482A1 PCT/MY2014/000048 MY2014000048W WO2014163482A1 WO 2014163482 A1 WO2014163482 A1 WO 2014163482A1 MY 2014000048 W MY2014000048 W MY 2014000048W WO 2014163482 A1 WO2014163482 A1 WO 2014163482A1
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WO
WIPO (PCT)
Prior art keywords
medium
protein
palm
fungi
agar
Prior art date
Application number
PCT/MY2014/000048
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English (en)
Inventor
Firdaus Othman MOHD
Original Assignee
Malaysian Palm Oil Board (Mpob)
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 Malaysian Palm Oil Board (Mpob) filed Critical Malaysian Palm Oil Board (Mpob)
Publication of WO2014163482A1 publication Critical patent/WO2014163482A1/fr

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Classifications

    • 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
    • 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

Definitions

  • the present invention relates to a medium for culturing fungi and yeast comprising a palm protein, monosaccharides and a bacteriological agar.
  • the palm protein is extracted from palm kernel meal.
  • this invention relates to a method of producing the medium for detection and enumeration of fungi and yeast.
  • Culture medium is a liquid or gel used to support the growth of microorganisms or cells.
  • Cell culture and microbiological culture are two major types of culture media.
  • the most common type of microbiological culture is an agar plate that contains agar and nutrients for routine cultivation of non-fastidious microorganisms such as bacteria and fungi. This kind of agar plate could support growth of different types of microbes, fungi and bacteria.
  • Potato dextrose agar (PDA) has been conventionally used in microbiological tests as a medium for fungi detection and enumeration. Protein extracted from potato is the major source of nutrient in this kind of culture medium.
  • PDA Potato dextrose agar
  • Protein extracted from potato is the major source of nutrient in this kind of culture medium.
  • Food has became expensive and scarce due to global exponential increment of human and animal populations. Consequently an alternative protein source is sought-after.
  • protein extracted from other plants such as legumes and wheat have great potential to be used as an alternative protein
  • the composition comprises between 4.5 to 5.5 g/1 of monobasic potassium phosphate, between 0.5 to 1.5 g/1 of ammonium chloride, between 0.5 to 1.5 g/1 of heptahydrate magnesium sulfate, between 30.0 and 50.0 g/1 of D(+) saccharose, and water.
  • Another Korea Patent No. 1020110079086 reveals a medium for culturing fungi, containing 2.5% of anhydroglucose, 1.8 weight % of yeast extract, 0.02% of bay salt, agar, and remaining amount of water.
  • the cultured fungi are Colletotrichum gloeosporioides.
  • the primary object of the present invention is to provide a culture medium called palm protein dextrose agar (PPDA) for detection and enumeration of fungi and yeast.
  • the culture medium is essentially made of palm protein, monosaccharides and bacteriological agar.
  • Another object of the present invention is to provide a non-selective culture medium for the growth of fungi and yeast.
  • the culture medium can be used to support the growth of Aspergillus oryzae, Rhizopus oryzae, Candida albicans, Aspergillus niger and Ganoderma sp.
  • Another object of the present invention is to provide a culture medium with a protein source obtained from palm kernel cake.
  • industrial byproducts such as palm kernel cake can be used as a starting material to obtain palm protein required as nutrient source in a culture medium.
  • Yet another object of the present invention is to provide a culture medium PPDA with comparable nutrient value to a conventional protein dextrose agar (PDA).
  • PDA protein dextrose agar
  • PPDA has a protein content between 4 to 8 %, fibre content between 0.1 to 0.3 %, fat content between 0.01 to 2 %, moisture content between 4.5 to 6.0 % and ash content between 2 to 3.5%.
  • the embodiment of the present invention describes a medium for microbiological culture comprising 2 to 20 g per litre of palm protein, 10 to 50 g per litre of monosaccharides, and 10 to 30 g per litre of bacteriological agar.
  • Yet another object of the present invention is to provide a method of producing a medium for microbiological culture comprising the steps of: mixing palm protein, monosaccharides, and bacteriological agar in distilled water to form a mixture; dissolving the mixture by stirring and boiling; and autoclaving the mixture at 105 to 120 °C to obtain a gel medium.
  • the present invention discloses a medium for microbiological culture comprising 2 to 20 g per litre of palm protein, 10 to 50 g per litre of monosaccharides, and 10 to 30 g per litre of bacteriological agar.
  • Color additives can be added in the culture medium for easy enumeration and clear observation on the growth of fungi. Further, color additives helps in differentiation between various species of fungi.
  • the preferred color additive is Congo Red C.I. 22120. However, color additives such as Remazol Brilliant Blue R can also be used.
  • the microbiological culture medium is a non-selective medium. It generally contains nutrient that most microorganisms need for growth and maintenance. Non-selective medium facilitates the increase in concentration of fungi and yeast to detectable levels. Hence the medium can be used to grow Aspergillus oryzae, Rhizopus oryzae, Aspergillus niger, Candida albicans or Ganoderma sp..
  • the microbiological culture medium has a final pH in a range of 4.5 to 6.5. Workability of a culture medium is essentially influence by the pH. In one embodiment, the growth of microorganisms is substantially slow in a culture medium of pH less than 4.5.
  • the pH of the culture medium can be controlled by adjustment with an acid or an alkali. Hydrochloric acid can be used as the acid, whereas sodium hydroxide can be used as the alkali.
  • Carbon compounds range from simple small molecules like sugars, organic acids and alcohol, to proteins, polysaccharides and lipids.
  • Glucose is preferably used as a carbon source in the present invention.
  • other monosaccharides such as fructose, galactose, xylose or ribose can also be used as a carbon source to sustain the growth of fungi in the culture medium.
  • Agar is chosen over gelatin as agar remains gel-like at growth temperatures. It is physically firmer and stronger than gelatin. Bacteriological agar acts as a solidifying agent to maintain the culture medium in a gel form during incubation.
  • the preferred bacteriological agar is extracted from algae Gelidium.
  • bacteriological agar extracted from the same class such as Ptewcladia and Gracilaria can also be used.
  • the culture medium could remain firm in a storage temperature as low as 4 °C up to 3 months.
  • bacteriological agar is generally resistant to breakdown caused by bacterial enzymes.
  • Palm protein is essentially used as a nitrogen or amino acid source to sustain the growth of fungi.
  • the palm protein is extracted from palm kernel meal, a solid residue obtained after extraction of palm kernel oil from palm fruits.
  • the preferred palm kernel meal generally contains about 20 wt % protein that can be extracted and used as starting material in manufacture of culture medium.
  • Palm protein is preferably extracted by an infusion of palm kernel meal with water, followed by an organic acid precipitation process.
  • the palm kernel meal is soaked in water to form a mixture. Then the mixture is preferably heated to obtain a protein- water supernatant. Palm protein is essentially extracted at this stage and be separated from water with an organic solvent.
  • the preferred organic solvent is ethanol as it is highly water miscible and highly volatile to facilitate drying.
  • a method of producing a medium for microbiological culture comprising the steps of: mixing palm protein, monosaccharides, and bacteriological agar in distilled water to form a mixture; dissolving the mixture by stirring and boiling; and autoclaving the mixture at 105 to 120 °C to obtain a gel medium.
  • the palm protein is mixed with distilled water, monosaccharides and a bacteriological agar to form palm protein dextrose agar (PPDA).
  • PPDA palm protein dextrose agar
  • the mixture is autoclaved to destruct all forms of life in the mixture.
  • Proximate analysis of the medium shows that the medium is having palm protein content between 4 to 8%, fiber content between 0.1 to 0.3%, fat content between 0.01 to 2%, moisture content between 4.5 to 6% and ash content between 2 to 3.5%. Palm protein content was determined according to Kjeldahl method and each sample was calculated by multiplying the nitrogen content with a factor of 6.25. The fat content was determined by ether extract method. The fibre content was determined according to Weende method.
  • Incubation of fungi is used to monitor the applicability of the resulting culture medium.
  • One cm 3 mat of Aspergillus oryzae ATCC® 10124, Rhizopus oryzae EPKi, Aspergillus niger ATCC® 10574 and Ganoderma sp. stock cultures are individually cut and be placed into individual PPDA.
  • the incubation periods for Aspergillus oryzae ATCC®10124, Rhizopus oryzae EPKi, and Aspergillus niger ATCC®10574 are 7 days.
  • the incubation period for Ganoderma sp. is preferably 12 days.
  • Candida albicans ATCC® 10231 is streaked onto an individual PPDA plate.
  • the incubation period for Candida albicans ATCC® 10231 is preferably 48 hours. A shorter incubation period is preferred for culturing of Candida albicans as the growing rate of yeast is faster than fungi. Consequently, all of the above mentioned microorganisms showed positive growth within the incubation period in the PPDA of the present invention.

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  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Health & Medical Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Genetics & Genomics (AREA)
  • Biotechnology (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Mycology (AREA)
  • Microbiology (AREA)
  • Biomedical Technology (AREA)
  • Botany (AREA)
  • Virology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

Cette invention concerne un milieu pour culture microbiologique comprenant de 2 à 20 g par litre de protéine de palme, de 10 à 50 g par litre de monosaccharides, et de 10 à 30 g par litre de gélose bactériologique.
PCT/MY2014/000048 2013-04-04 2014-04-03 Gélose dextrosée à la protéine de palme (ppda) pour détecter et énumérer les champignons et la levure WO2014163482A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
MYPI2013700524 2013-04-04
MYPI2013700524 2013-04-04

Publications (1)

Publication Number Publication Date
WO2014163482A1 true WO2014163482A1 (fr) 2014-10-09

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
NL2033340B1 (en) * 2022-10-18 2023-06-19 Yunnan Tobacco Company Kunming Branch Trichosporon asahii and application thereof

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5756303A (en) * 1995-02-20 1998-05-26 Sapporo Breweries Limited Culture medium and a microbiological test method employing the same
KR20110079086A (ko) * 2009-12-31 2011-07-07 대한민국(농촌진흥청장) 진균 배양용 배지
US8313938B1 (en) * 2004-04-26 2012-11-20 Hardy Diagnostics Culture medium for cultivation of microorganisms

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5756303A (en) * 1995-02-20 1998-05-26 Sapporo Breweries Limited Culture medium and a microbiological test method employing the same
US8313938B1 (en) * 2004-04-26 2012-11-20 Hardy Diagnostics Culture medium for cultivation of microorganisms
KR20110079086A (ko) * 2009-12-31 2011-07-07 대한민국(농촌진흥청장) 진균 배양용 배지

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
ACUMEDIA, AGAR, BACTERIOLOGICAL (7178, 4 March 2012 (2012-03-04) *
MOHD FIRDAUS OTHMAN: "Palm protein dextrose agar (PPDA): a new medium for culturing yeasts and fungi", MALAYSIAN PALM OIL BOARD, MALAYSIA *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
NL2033340B1 (en) * 2022-10-18 2023-06-19 Yunnan Tobacco Company Kunming Branch Trichosporon asahii and application thereof

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