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Contains: - fermentation tests Tetragenococcus halophila - decarboxylation tests - detection killer yeast and killer toxins - yeast identification - YNB composition - histamine and tyramine production in media Pediococcus halophilus Fermentatie testen (Uchida'82) Medium: Per liter: 2 g Yeast Extract 10 g Poly peptone 5 g KH2PO4 50 g NaCl 0.04 g bromocresol purple 10 g test carbohydrate
pH 6.8
2 ml in kleine tubes (10 x 105 mm). Inoculatie met 0.05 ml broth, incubatie 30oC, 5 dagen. Test-carbohydraten, behalve arabinose, kunnen worden gesterilizeerd met de andere ingredienten. Sterilisatie 10 min 121oC. Als voorculture: glucose 0.2%. Test-carbohydraten: arabinose, lactose, melibiose, sorbitol, mannitol, xylose, mannose, sucrose, raffinose, trehalose. Ammonium van arginine (Abstracts of Micr. Methods '63 (a0111BR), ed. VBD Sherman, Wiley-Interscience) Medium: Per Liter: 5 g Yeast Extract 5 g tryptone 2 g K2HPO4 0.5 g glucose 3 g arginine (50 g NaCl)
pH 7.0
3 druppels medium + 1 druppel Nessler reagent. Bij ammonia geeft dit een groene kleur.
culturing (kecap
October 19, 2001
Protocol
Molecular Cell Physiology
Kecap
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Decarboxylatie van aminozuren Medium: Per Liter: 5 g bactopepton 3 g yeast extract 1 g glucose 20 g aminozuur (phe, tyr, his, asp) 1 ml (?) bromocresol purple (1.6%) (50 g NaCl)
pH 6.7-6.8
Door fermentatie wordt de kleur geel, door de decarboxylatie wordt de kleur paars weer
culturing (kecap
October 19, 2001
Protocol
Molecular Cell Physiology
Kecap
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Detection of Killer yeasts and killer activity From: Kagiyama S, Aiba T, Kadowaki K and Mogi K, New Killer Toxins of Halophilic Hansenula anomala. Agric. Biol. Chem. 52 (1), 1 - 7, 1988.
Plates: glucose 2%, pepton 2%, yeast extract 1%, (NaCl 8%,) methylene blue 0.03%, agar 2%0.1 M citrate phosphate buffer, pH 4.8. Detection of killer yeasts The detection is performed by the cross-streak test. A target strain was inoculated on the plates and then the strain to be tested for killing activity was cross-streaked on them, followed by incubation at 23oC for 4 days. If the strain has killer activity then the target cells become blue due to methylene blue staining. Detection of killer yeasts Killer yeast cultures are filtered through nitrocellulose membrane filters. 0.1 ml portions of the filtrates were pipetted into cups on the plates, with 8% NaCl, each inoculated with cells of the test strain. The plates were incubated at 23oC for 4 days. The diameter of the inhibitory zone around each cup was measured and the area of the inhibitory zone was calculated. One unit is the amount of killer toxin that inhibits one square cm of the test strain.
culturing (kecap
October 19, 2001
Protocol
Molecular Cell Physiology
Kecap
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Identification of yeasts From: Mori H. , Toriumi H.1985. New method for selective counting of salt-tolerant yeasts in shoyu mashes. Hakkokogaku Kaishi 63, 47 - 54.
Plates: - with 15% NaCl, pH 5.2 or pH 6.5, with or without 1/15M LiCl. Z.rouxii: only grows on medium with pH 5.2 and without Acid Phosphatase (ACP) activity Candida (III): No growth on pH 6.5, but grows with LiCl, has ACP activity. Candida (V): which grows on all media except pH 6.5 with LiCl and has no ACPactivity Pichia/Debaryomyces: no growth on pH 6.5 with LiCl, has ACP-activity Candida (IV): grows on all media and has ACP-activity Hansenula: no growth on LiCl media, has ACP activity ACP-activity determination: Reagents 0.01 M p-nitrophenyl phosphate 0.04 M glycine-NaOH, buffer pH 10.5 or acetate buffer (0.05 M) with 50 mg procedure Mix a suspension of resting cells with a equal volume (0.3 ml) of p-nitrophenyl phosphate and incubate it for a maximum of 6 h at 37oC. When ACP is present a yellow colour will develop on addition of 0.3 ml of glycine-NaOH buffer.
culturing (kecap
October 19, 2001
Protocol
Molecular Cell Physiology
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Media Composition of Yeast Nitrogen Base without amino acids (6.7 g/l):
N-source:
(NH4)2SO4
Salts:
Potassium phosphate Magnesium sulphate Sodium chloride Calcium chloride Borate Copper sulphate Potassium iodide Ferric chloride Manganous sulphate Sodium molybdate Zinc sulphate Biotine Ca-pantothenate Folinic acid Inositol Niacine Aminobenzoeenic acid Pyridoxine HCl Riboflavine Thiamine HCl
Traces:
Vitamines:
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g/l 5
µg/l
1 0.5 0.1 0.1 500 40 100 200 400 200 400 2 400 2 2000 400 200 400 200 400
October 19, 2001
Protocol
Molecular Cell Physiology
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Determination of histamine (Rice, S.L. and Koehler, P.E: J. Milk Food Technol. 39 (3), 166-169 (1976)) Medium: - decarboxytlase assay medium: 10 g Tryptone 10 g Yeast Extract 10 g Glucose 0.1 g tyrosine 0.1 g histidine per liter - analysis - 1 ml medium mixen met 4.0 ml 0.4 N HClO4 - 2 ml van de mixture overbrengen naar een centrifuge buis met 5 ml nbutanol:chloroform (3:2 v/v), 0.25 ml 5 N NaOH en 0.75 g NaCl - 5 min. schudden, + centrifugeren (vrij histidine verwijderen) - butanol:chloroform naar tube met 2.5 NaCl verzadigde 0.1 N NaOH, 1 min. schudden en centrifugeren - 4 ml naar 3e tube met 2.5 ml 0.1 N HCl en 7.5 ml n-heptane, 1 min schudden, centrifugeren, de organische fase verwijderen via aspiratie - Toevoegen aan 2 ml 0.4 ml 1 N NaOH + 0.1 ml 0-phtalaldehyde (OPT) (10mg/ml in methanol). Na 4 min, 0.2 ml 3 N HCl toevoegen. - Fluorenscentie op 450 nm, als gevolg van activatie op 360 nm, meten met een Spectrofluormeter. Standaard van 1 µg/ml histamine. Tyramine bepaling - 5 ml pH 10.0 stellen met vaste Na2CO3. - NaCL verzadigen (ong. 1.5 g) en 3.75 ml n-butanol toevoegen. 10 min schudden
culturing (kecap
October 19, 2001
Protocol
Molecular Cell Physiology
