Kecap Page 1 of 6. Test-carbohydraten: arabinose, lactose, melibiose, sorbitol, mannitol, xylose, mannose, sucrose, raffinose, trehalose.

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1 Kecap Page 1 of 6 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) 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 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) Per Liter: 5 g Yeast Extract 5 g tryptone 2 g K2HPO4 0.5 g glucose 3 g arginine ph 7.0 (50 g NaCl) 3 druppels medium + 1 druppel Nessler reagent. Bij ammonia geeft dit een groene kleur.

2 Kecap Page 2 of 6 Decarboxylatie van aminozuren 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%) ph (50 g NaCl) Door fermentatie wordt de kleur geel, door de decarboxylatie wordt de kleur paars weer

3 Kecap Page 3 of 6 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, 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 23 o C 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 23 o C 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.

4 Kecap Page 4 of 6 Identification of yeasts From: Mori H., Toriumi H New method for selective counting of salt-tolerant yeasts in shoyu mashes. Hakkokogaku Kaishi 63, 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 37 o C. When ACP is present a yellow colour will develop on addition of 0.3 ml of glycine-naoh buffer.

5 Kecap Page 5 of 6 Media Composition of Yeast Nitrogen Base without amino acids (6.7 g/l): g/l µg/l N-source: (NH 4 ) 2 SO 4 5 Salts: Potassium phosphate 1 Magnesium sulphate 0.5 Sodium chloride 0.1 Calcium chloride 0.1 Traces: Borate 500 Copper sulphate 40 Potassium iodide 100 Ferric chloride 200 Manganous sulphate 400 Sodium molybdate 200 Zinc sulphate 400 Vitamines: Biotine 2 Ca-pantothenate 400 Folinic acid 2 Inositol 2000 Niacine 400 Aminobenzoeenic acid 200 Pyridoxine HCl 400 Riboflavine 200 Thiamine HCl 400

6 Kecap Page 6 of 6 Determination of histamine (Rice, S.L. and Koehler, P.E: J. Milk Food Technol. 39 (3), (1976)) - 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 n- butanol: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 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