Proizvodnja hidrolitičkih enzima fermentacijom na poljoprivrednom otpadu pomoću različitih vrsta iz roda Bacillus

During the production and processing of food, large quantities of waste are generated which need to be properly stored and, at best, found new use and utilized. An extremely large problem worldwide is certainly the large amount of agroindustrial waste that is generated on a daily and especially annual basis. If agroindustrial waste is directly stored in the environment, it causes a greenhouse effect and is a large-scale environmental problem. However, agroindustrial waste has recently been regarded as an extremely valuable raw material that can be converted into an added-value raw material by applying different biotechnological approaches. Accordingly, this doctoral dissertation deals with the global problem of utilization and quality improvement of various agro-industrial wastes through the application of biotechnology. Modification and improvement of quality is achieved by growing the production microorganism on a solid surface, that is, agroindustrial waste. This is a rather new method which involves the use of cheap materials but they usually contain large amount of protein, sugar and mineral as well as bioactive compounds such as polyphenols, carotenoids and dietary fibers. Accordingly, the first part of this doctoral dissertation is the selection of the microorganism producer. During the selection, a rich collection of various commercial and natural bacterial strains was utilized, which is available at the Department of Biochemical Engineering and Biotechnology, Faculty of Technology and Metallurgy. Various strains have been tested for the ability to produce α-amylase, cellulase, pectinase and protease using selective medium on agar plates. In this way, one of the Bacillus strains was found to be particularly prominent in the production of targeted enzymes. Due to the fact that this strain is a natural isolate, identification by sequencing 16SrRNA encoding gene and subsequent analysis of the obtained sequence using the BLAST (Basic Logical Alignment Search Tool) was performed. Further, the obtained and isolated strain (Bacillus sp. TMF-1) was used in the solid-state fermentation process with the main objective to examine the possibility of the selected microorganism to produce desired enzymes under the different substrate:water ratios by using different types of agroindustrial waste materials such as wheat bran, soybean meal, olive oil cake, sunflower meal for growth. Following the cultivation of microorganism producer and production of targeted enzymes, it has been found that agro-industrial waste can be transferred to an even better growing medium if a certain treatment of the raw material is carried out. Accordingly, the aforementioned agroindustrial wastes have been chemically treated by the use of acids and bases, the use of ultrasound as well as microwave technology and used for enzymes production by Bacillus sp. TMF-1.U toku proizvodnje i obrade hrane stvaraju se velike količine otpada koje je neophodno skladištiti na pravi način i u najboljem slučaju ponovo iskoristiti. Izuzetno veliki problem širom sveta je svakako stvaranje velikih količina agro-industrijskog otpada na dnevnom a naročito godišnjem nivou. Ukoliko se agro-industrijski otpad direktno skladišti u okolnu životnu sredinu nastaje efekat staklene bašte i to je ekološki problem velikih razmera. Međutim, agro-industrijski otpad je skoro klasifikovan kao izuzetno vredna sirovina koja se može prevesti u sirovinu sa dodatnom vrednošću primenom različitih biotehnoloških pristupa. Shodno tome, ova doktorska disertacija se bavi globalnim problemom iskorišćenja i unapređenja kvaliteta različitog agroindustrijskog otpada primenom biotehnologije. Modifikacija i unapređenje kvaliteta je postignuta gajenjem proizvodnog mikroorganizma na čvrstoj podlozi, tj. agro-industrijskom otpadu. Ovo je prilično nova metoda koja podrazumeva upotrebu jeftinih materijala koji sadrže velike količine proteina, šećera i minerala kao i bioaktivnih jedinjenja kao što su polifenoli, karotenoidi i dijetetska vlakna. Prvi deo ove doktorske disertacije je selekcija mikroorganizma producenta. Tokom ove selekcije upotrebljena je bogata kolekcija različitih prirodnih i komercijalnih sojeva bakterija dostupnih na Katedri za Biohemijsko inženjerstvo i biotehnologiju, Tehnološko-metalurškog fakulteta. Različiti sojevi su ispitani na sposobnost proizvodnje α-amilaza, celulaza, pektinaza i proteaza korišćenjem selektivnog medijuma na agar pločama. Na ovaj način utvrđeno je da jedan od Bacillus sojeva ima izuzetan potencijal za proizvodnju ciljanih enzima. Shodno činjenici da je ovaj soj prirodnog porekla, izvršena je identifikacija sekvencionisanjem gena za kodiranje 16SrRNA i naknadnom analizom dobijene sekvence korišćenjem osnovnog alata za pretraživanje logičkog poravnanja (na engl.’’ Basic Logical Alignment Search Tool’’, BLAST). Zatim, dobijeni i izolovani soj (Bacillus sp. TMF-1) je korišćen za fermentaciju na čvrstoj podlozi sa osnovnim ciljem da se ispita mogućnost odabranog mikroorganizma da proizvede željene enzime pri različitom odnosu supstrat:voda korišćenjem za rast različitog agro-industrijskog otpadnog materijala kao što su: pšenične mekinje, sojina sačma, pogača nakon ceđenja maslinovog ulja, suncokretova sačma. Praćenjem gajenja mikroorganizma producenta i proizvodnje ciljanih enzima, utvrđeno je da se agroindustrijski otpad može prevesti u još bolji medijum za rast ukoliko se sirovine podvrgnu određenim pretretmanima. Prema tome, pomenuti agro-industrijski otpadi su pretretirani hemijski korišćenjem kiselina i baza ali i primenom tehnologije ultrazvuka i mikrotalasa i korišćeni za proizvodnju enzima Bacillus sp. TMF-1-om