Isolation and identification of potentially toxigenic, filamentous fungi and detection of economically-important mycotoxins in locally produced soybean curd

The mean viable counts of putative filamentous fungi (molds) from tokwa and taho samples sold at selected areas in Sta. Cruz and Calamba City, Laguna [Philippines] were 4.20 x 10 sup 1 CFU/g and 1 EAPC (estimated aerobic plate count), and 3.73 x 10 sup 3 CFU/g and 1 EAPC, respectively. Twenty mold isolates were isolated from 57 pooled tofu samples (39 tokwa and 18 taho). Consensus analyses of ITS rDNA sequences revealed that fifteen of the isolates (SA3, 4, 7-12, 14, 16, 19-23) were identified as Geotrichum candidum (99 to 100% sequence similarity), one (SA2) had probable identity of Aspergillus flavus or A. oryzae (99% similarity), one isolate each of Trichoderma reasei (SA26) (100% similarity), Aspergillus sydowii (CB37) (100% similarity, Arthrinium sacchari (CB39) (99% similarity) and Saparochaete sp. (CB21) (87% similarity). Protein analysis using MALDI-TOF also showed that SA7, 16, 19 and 21 had probable identity of G. caddidum or G. klehbanii with percent identity of 79.4, 83.7, 77.5 and 75.8 respectively. Cultural and morphological characterization of the six representative isolates (SA2, SA16, SA26, CB21, CB37 and CB39) revealed that isolates, except CB39, conformed to the molecular identity provided by ITS rDNA sequence analyses. SA2 shared similarities with both A, flavus and A. oryzae though it appeared to be more closely similar to A. flavus due to its dark green conidia which retained its color until the 14th day of incubation. SA16 and SA26 shared similar characteristics with Geotrichum candidum and with T. reesei, respectively. CB21 shared characteristics with both Saprochaete clavata and Magmusiomyces capatatum while CB37 was silimar to A. sydowii, CB39, however, did not share most of characteristics of A. sacchari. Two out of the twenty (10%) mold isolates were potentially toxigenic (SA2 as A. Flavus or A. oryzae and SA26 as T. reesei). The sample prevalence was, thus, 3.50% from 2 out of 57 pooled samples. Isolate SA2 was obtained from tokwa while SA26 was isolated from taho. When aflatoxin B1 (AFB1) ochratoxin A (OTA) and fumonisin (FUM) were assayed for and quantified in tokwa and taho using competitive ELISA, four tokwa samples and one taho sample contained AFB at concentrations ranging from 8.33 to 14.06 ppb which are within the acceptable limit of 20 ppb set by the Philippine National Standard. None of samples were detected with OTA. Two tokwa and three taho samples contained FUM; of those, one tokwa and one taho samples had concentrations (613ppb and 6533 ppb, respectively) which exceeded internationally established FUM limits for food products (4000 ppb). In the preliminary investigation in the in vitro biological detoxification of FUM using live and heat-killed preparation of Saccharomyces cerevisiae (PNCM Accession No. 2025), FUM was no longer detected in the supernatant and pellet recovered from the soymilk initially spiked with FUM (at an initial concentration of 6 ppm) and inoculated with live cells. However, FUM was recovered at a concentration of 6.61 not equal to 0.33 ppm in the supernatant from the soymilk inoculated with heat-killed cells, but not in the pellet.