Apinya Assavanig

Isolation, characterization and function of laccase from Trichoderma

Of fourteen natural isolates of Trichoderma, no correlation was found between substrate weight loss and phenol oxidase (PO) activity in rice straw cultures. The highest PO producer from these laccase-positive strains was subjected to UV mutagenesis in order to select high and low PO activity mutants. There was no significant difference in substrate weight loss for mutant strains with six times higher and six times lower PO activity than the parent strain. Nor did the enzyme activity result in decreased growth inhibition by inhibitory phenolic compounds. PO enzyme from the parent Trichoderma and one of its high-PO-activity mutants was subsequently purified by ethanol precipitation from liquid cultures optimized by supplementation with copper sulphate and cycloheximide. Protein staining and activity staining of disc electrophoresis gels showed that only one PO enzyme of approximately 71 000 Da was produced. The enzyme could be defined as a laccase (benzenediol: oxygen oxidoreductase E.C. 1.10.3.2) because it catalysed the oxidation of syringaldazine and p-phenylenediamine in the absence of hydrogen peroxide, and because it was inhibited by cetyltrimethylammonium because but not by cinnamic acid. No specific in-vivo function could be assigned to this enzyme.

Co-culturing of Pichia guilliermondii enhanced volatile flavor compound formation by Zygosaccharomyces rouxii in the model system of Thai soy sauce fermentation.

The roles of salt-tolerant yeasts such as Zygosaccharomyces rouxii, Candida versatilis, and Candida etchellsii in the production of volatile flavor compounds (VFCs) in soy sauce fermentation have been well documented. However, the knowledge of VFC production by other salt-tolerant yeasts is still limited. In this work, the roles of Z. rouxii and Pichia guilliermondii strains in VFC production were investigated in moromi medium as a model system for soy sauce fermentation. Inoculation of a single culture of either Z. rouxii or P. guilliermondii as well as co-cultures of these two yeasts into moromi medium showed increased numbers of viable yeast at around 0.7 to 1.9 log CFU/mL after 7days of cultivation at 30°C. During cultivation, both single and co-cultures displayed survival over a 7-day time period, compared with the controls (no culture added). Overall, yeast inoculation enhanced the production of VFCs in the moromi media with higher amounts of ethanol, alcohols, furanones, esters, aldehyde, acid, pyrone and phenols, known as important characteristic flavor compounds in soy sauce. Moreover, the co-culture produced more alcohols, furanones, esters, maltol and benzoic acid than the single culture of Z. rouxii.

Development and Assessment of a Real-Time PCR Assay for Rapid and Sensitive Detection of a Novel Thermotolerant Bacterium, Lactobacillus thermotolerans, in Chicken Feces

ABSTRACT A new real-time PCR assay was successfully developed using a TaqMan fluorescence probe for specific detection and enumeration of a novel bacterium, Lactobacillus thermotolerans, in chicken feces. The specific primers and probe were designed based on the L. thermotolerans 16S rRNA gene sequences, and these sequences were compared to those of all available 16S rRNA genes in the GenBank database. The assay, targeting 16S rRNA gene, was evaluated using DNA from a pure culture of L. thermotolerans, DNA from the closely related bacteria Lactobacillus mucosae DSM 13345T and Lactobacillus fermentum JCM 1173T, and DNA from other lactic acid bacteria in quantitative experiments. Serial dilutions of L. thermotolerans DNA were used as external standards for calibration. The minimum detection limit of this technique was 1.84 × 103 cells/ml of an L. thermotolerans pure culture. The assay was then applied to chicken feces in two different trials. In the first trial, the cell population was 104 cells/g feces on day 4 and 105 cells/g feces on days 11 to 18. However, cell populations of 106 to 107 cells/g feces were detected in the second trial. The total bacterial count, measured by 4′,6-diamidino-2-phenylindole (DAPI) staining, was approximately 1011 cells/g feces. These results suggest that in general, L. thermotolerans is a normal member of the chicken gut microbiota, although it is present at relatively low levels in the feces.

Influence of minced pork and rind ratios on physico-chemical and sensory quality of Nham - a Thai fermented pork sausage.

The effects of incorporating varying levels of minced pork and rind on physico-chemical and sensory quality of Nham were studied. An increase in cooked pork rind resulted in higher moisture, lipid, and initial pH values of Nham (P<0.05). However, no significant effects were observed on fermentation characteristics of Nham (P>0.05). At the end of fermentation, Nham with a higher meat component exhibited higher texture profile analysis force, hardness, and cohesiveness (P<0.05). The results suggested the importance of meat on the restructuring effect, which contributes to the texture formation of Nham. Incorporation of a higher amount of cooked pork rind improved water-binding properties, leading to decreased weight loss and released water. Based on the results of sensory evaluation, up to 43% pork rind can be used in the formulation with no adverse effect on texture and overall liking of Nham. However, the ratio of 5:5 was the most appropriate for minimising the cost of production.

Technology transfer for small and medium soy sauce fermentation factories in Thailand : a consortium approach

Abstract There are a number of soybean fermentation products in Thailand. These include soy sauce, soybean paste, fermented soybean curd and fermented black soybeans. Although there are two or three large factories, the soybean fermentation industry mainly comprises of small and medium size factories. The manufacturing techniques employed by these small factories are usually those passed from generation to generation with little or no improvement. Due to the lack of qualified personnel and low financial investment, there are minimal research and development activities by either large or small factories. The lack of research and development activity greatly inhibits the capability of these factories to improve either their production technology or the quality of their products. A few years ago, a small research and development unit was set up through the support of the Thailand Research Fund (TRF) and a consortium of small and medium size soy sauce manufactures. This unit was called the Quality Control and Training Center for Soybean Fermentation (QCTC-Soybean Fermentation). By working closely with the consortium, this unit has initiated research and development activities that have benefited members of the consortium. The objectives of the QCTC-Soybean Fermentation program have been to improve the quality of soybean fermentation products and to improve manufacturing techniques. Some examples of QCTC-achievements include the introduction of improved microbial inocula (Aspergillus oryzae, Zygosaccharomyces rouxii and Tetragenococcus halophilus) for soy sauce fermentation, the introduction of new technique for preparing koji, the use of large fiberglass tanks instead of small earthenware containers for second stage soy sauce fermentation, and the introduction of cost effective waste treatment systems. These inputs have helped the small industry consortium members to improve the quality of their products and/or to reduce production costs.

Isolation and characterization of acid-sensitive Lactobacillus plantarum with application as starter culture for Nham production.

The aim of this study was to derive new starter culture variants that are unable to grow below pH 4.6, the desirable pH of the Thai fermented pork sausage, Nham, specified by Thailand Food Standard, and apply them in Nham fermentation. Several acid-sensitive mutants of one of the commercial Nham starter cultures, Lactobacillus plantarum BCC 9546, were isolated as spontaneous neomycin-resistant mutants. The growth of three representative mutants was characterized in MRS broth, which revealed that their cell numbers and acid production were lower than that of the wild-type. The H(+)-ATPase activities of the three mutants were found significantly lower than that of the wild-type under either neutral or acidic conditions. Consequently, internal pH values of the mutants appeared to be lower, especially in acidic environment (pH 5). The most acid-sensitive mutant was applied in experimental Nham production and the pH of Nham fermented with the mutant had significantly higher pH at the end of fermentation (3 days) and after an additional 4 days of storage at 30 degrees C. These results indicate that the use of acid-sensitive L. plantarum as starter culture can reduce the severity of post-acidification and increase the shelf life of Nham at ambient temperature.

Glutaminase-producing Meyerozyma (Pichia) guilliermondii isolated from Thai soy sauce fermentation

In this study, 34 yeast isolates were obtained from koji and moromi samples of Thai soy sauce fermentation. However, the most interesting yeast strain was isolated from the enriched 2 month-old (M2) moromi sample and identified as Meyerozyma (Pichia) guilliermondii EM2Y61. This strain is a salt-tolerant yeast that could tolerate up to 20% (w/v) NaCl and produce extracellular and cell-bound glutaminases. Interestingly, its glutaminases were more active in 18% (w/v) NaCl which is a salt concentration in moromi. The extracellular glutaminases activity was found to be much higher than that of cell-bound glutaminase. The highest specific activity and stability of the extracellular glutaminase were found in 18% (w/v) NaCl at pH4.5 and 37°C. A challenge test by adding partially-purified extracellular glutaminase from M. guilliermondii EM2Y61 into 1 month-old (M1) moromi sample showed an increased conversion of L-glutamine to L-glutamic acid. This is the first report of glutaminase producing M. guilliermondii isolated from the moromi of Thai soy sauce fermentation. The results suggested the potential application of M. guilliermondii EM2Y61 as starter yeast culture to increase l-glutamic acid during soy sauce fermentation.

Chemical and Organoleptic Comparison of Fish Sauce Made from Cold Water Species and Typical Thai Fish Sauce

ABSTRACT Organoleptic and chemical properties of fish sauce made experimentally from the four cold water fish species–Arctic capelin, Atlantic herring, Atlantic cod and blue whiting–were compared with three first grade commercial fish sauces made from tropical anchovies. Although the flavor of the fish sauces made from cold water species were judged as inferior to the flavor of the tropical fish sauces, the blue whiting and Atlantic herring sauces obtained fair ranking. Chemical analyses revealed that sugar had been added in all the three commercial sauces. Although sugar is added during blending just before bottling, flavor enhancing chemical reactions, between amino groups and reducing sugars, probably occur during storage of the final product. Accordingly, this procedure should be adapted also in preparation of fish sauce from cold water species.