Research On Isolation And Identification Of Patulin-Producing Strains And Their Growth Metabolic Characteristcs

Patulin(Pat) is a kind of secondary metabolite of fungal toxins, which is teratogenic, carcinogenic and mutagenic to animals. Since joining the WTO, the patulin residue has become an important factor of affecting apple juice quality and safety, also limiting Chinese apple juice exports. So effectively controlling and reducing the patulin concentration in apple products has become a hot spot in domestic and international studying. Patulin in apple products is mainly from the contamination of patulin-producing strains. Although people have made some success on patulin controlling techniques in apple juice production and processing, we haven't radically resolve the patulin problem. So we must do more in-depth and extensive research on inhibiting patulin-producing strains growth and producing patulin. In this paper, patulin-producing strains were isolated from apples which are different types and from different areas. By morphological and ITS sequences analysis, patulin-producing strains were identified and their phylogenetic relationships were also known in their phylogenetic tree. After knowing their reliable biological information, the metabolic patulin characteristics and kinetic models of main patulin-producing strains were studied, especially the high levels of patulin producing strain, so as to provide a theoretical basis for patulin controlling from the source of apple.The main results in this paper are as follows:(1) 10 patulin-producing strains were isolated from storage apples.(2) By morphological characteristics we concluded strain 1 and 8 are Aspergillus, and the remaining eight strains are Penicillium. By ITS sequences analysis we got reliable results: strain 1 Aspergillus sydowii,2 Penicillium paneum,3 Penicillium griseofulvum,4 Penicillium chrysogenum,5 Penicillium expansum,6 Penicillium expansum,7 Penicillium chrysogenum,8 Aspergillus oryzae,9 Penicillium expansum,10 Penicillium expansum。(3) Phylogenetic tree showed that 10 patulin—producing strains from storage apples have different levels of the phylogenetic relationship.(4) By analyzing the relationship between the main strains and patulin production, we know Penicillium is the main contamination fungi that result in patulin problem in apples. (5) For different strains, shaking or not has different effect on patulin production. No shaking is more favorable to most strains producing patulin. Also different culture medium is very important to patulin production, and especially addition Mn²⁺ can increase the patulin production.(6) The optimizing condition On the base yeast extract liquid culture medium for patulin production condition of Penicillium griseofulvum was formulated vas Placket -Barman design and response surface analysis method (RSM). The optimum condition is: glucose 75.86g/L, inoculation volume 0.92%, MnCl₂·4H₂O 0.03g/L, yeast extract 1.0g/L, peptone 1.0g/L, pH value naturally, medium volume 50mL/250mL, temperature 25℃. Under the condition the maximum production of Penicillium griseofulvum is 528.25mg/L.(7) By 4 factors of quadratic orthogonal rotary combination tests, we knew inoculation volume had significant effect on patulin production of Penicillium griseofulvum in apple culture medium, and temperature, soluble solid, pH value had no significant effect. The optimizing condition was gotten: soluble solid concentration 10.86%,inoculation 0.92%,pH value 4.7, temperature 25℃. Under the condition the maximum production of Penicillium griseofulvum in apple culture medium is 483.79mg/L。(8) Penicillium griseofulvum fermentation dynamics model was constructed both in optimizing experiment culture medium and apple medium, which had the similar kinetic growth, product and substrate consumption characteristics, so the same fermentation models were used. The kinetic models provided a reasonable description for growth, product and substrate consumption variation along with the fermentation process. So these models can provide the theoretical support for predicting and controlling this strain growth and producing patulin.