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dc.contributor.advisorPejin, Jelena
dc.contributor.otherŠćiban, Marina
dc.contributor.otherPejin, Jelena
dc.contributor.otherDimić, Gordana
dc.contributor.otherMojović, Ljiljana
dc.contributor.otherĐukić-Vuković, Aleksandra
dc.creatorRadosavljević, Miloš
dc.date.issued2017-05-31
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dc.identifier.urihttp://www.cris.uns.ac.rs/DownloadFileServlet/IzvestajKomisije148836827211238.pdf?controlNumber=(BISIS)104282&fileName=148836827211238.pdf&id=7276&source=NaRDuS&language=srsr
dc.description.abstractPivski trop čini približno 85% od ukupnih sporednih proizvoda proizvodnje piva, i dostupan je po veoma niskim cenama tokom čitave godine. Pivski trop ima veliku perspektivu za primenu u biotehnologiji i proizvodnji visoko vrednih proizvoda. Jedna od veoma ekološki i ekonomski isplativih alternativa je upotreba pivskog tropa u proizvodnji mlečne kiseline, jer se poslednjih par decenija uočava intenzivan rast potražnje za mlečnom kiselinom. Mlečna kiselina je najvažnija hidroksikarbonska kiselina široko rasprostranjena u prirodi, sa velikom primenom u prehrambenoj, farmaceutskoj, tekstilnoj i hemijskoj industriji i industriji prerade kože. Cilj istraživanja ove doktorske disertacije je ispitivanje primene pivskog tropa u proizvodnji mlečne kiseline. Prvo je izvršena optimizacija enzimske hidrolize pivskog tropa u cilju dobijanja što je moguće veće koncentacije redukujućih šećera neophodne za mlečno-kiselu fermentaciju. Hidrolizat pivskog tropa je dobijen enzimskom hidrolizom dodatkom komercijalnih enzima za razgradnju skroba i celuloze. Parametri čiji je uticaj na efikasnost enzimske hidrolize ispitanu su: pH vrednost, temperatura hidrolize i količina dodatih enzima. Nakon što su određeni najbolji uslovi razgranje pivskog tropa, dobijeni postupak hidrolize je primenjen u proizvodnji hidrolizata pivskog tropa koji je korišćen u mlečno-kiselim fermentacijama. Nakon toga je ispitana mlečno-kisela fermentacija sa dva proizvodna mikoorganizma. Kao proizvodni mikroorganizmi u mlečno-kiselim fermentacijama primenjena su dva soja bakterija mlečne kiseline: Lactobacillus fermentum PL-1 i Lactobacillus rhamnosus ATCC 7469. Ispitan je uticaj dodatka različitih koncentracija ekstrakta kvasca (0,5-5,0%) uz korekciju pH vrednosti tokom fermentacije sa dodatkom kalcijum-karbonata. U zavisnosti od udela L-(+)- i D-(-)-mlečne kiseline koje nastaju tokom fermentacije izabran je proizvodni mikroorganizam koji proizvodi više L-(+)-mlečne kiseline. U daljim ispitivanjima je ispitan uticaj korekcije pH pomoću natrijum-hidroksida kao i dodatak različitih koncentracija ekstrakta kvasca (0,5-5,0%) i redukujućih šećera (2,7; 5,4 i 8,1%) u hidrolizatu pivskog tropa na mlečno-kiselu fermentaciju pomoću odabranog soja bakterija mlečne kiseline. Na osnovu dobijenih rezultata izabrana je najbolja koncentracija redukujućih šećera i ekstrakta kvasca koji će se koristiti u daljim istraživanjima. Takođe je ispitana i mogućnost zamene skupog ekstrakta kvasca i glukoze sa obnovljivim sirovinama, kao što su pivski kvasac, džibra i bistra džibra. Ispitan je uticaj dodatka različitih koncentracija pivskog kvasca (0,5-5,0%), džibre (5-20%) i bistre džibre (5-50%) pre fermentacije kao i dodatak bistre džibre u dolivnoj fermentaciji, na mlečno-kiselu fermentaciju hidrolizata pivskog tropa. Ispitan je i dolivni postupak fermentacije hidrolizata pivskog tropa dodatkom glukoze, glukoze i ekstrakta kvasca i sladovine. Takođe je ispitana mogućnost izvođenja više uzastopnih fermentacija sa imobilisanim ćelijama odabranog soja bakterija mlečne kiseline u kalcijum-alginatu. Na osnovu eksperimentalnih rezultata zaključujeno je da je dodatak kalcijum-karbonata imao pozitivan uticaj na proizvodnju mlečne kiseline sa L. fermentum i L. rhamnosus. Sa dodatkom kalcijum-karbonata povećali su se utrošak redukujućih šećera, koncentracija i prinos mlečne kiseline i vijabilnost ćelija L. fermentum i L. rhamnosus. Ekstrakt kvasca i kalcijum-karbonat su imali značajan uticaj na proizvodnju mlečne kiseline sa L. fermentum i L. rhamnosus. U fermentacijama sa L. fermentum najveći prinos ukupne mlečne kiseline (44%) je postignut sa dodatkom 5,0% ekstrakta kvasca i 2,0% kalcijum-karbonata. U fermentacijama sa L. rhamnosus najveći prinos ukupne mlečne kiseline (98%) i L-(+)-mlečne kiseline (96%) je ostvaren u fermentaciji sa dodatkom 2,0% ekstrakta kvasca i 2,0% kalcijum-karbonata. Na osnovu rezultata odlučeno je da se u daljim ispitivanjima mlečno-kisele fermentacije hidrolizata pivskog tropa kao proizvodni mikoorganizam koristi L. rhamnosus. Primenom natrijum-hidroksida za korekciju pH je skratila fermentaciju za 48 sati a ostvareno je i značajno povećanje zapreminske produktivnosti L-(+)-mlečne kiseline (za 200%, povećanje sa 0,21 na 0,63 g/l·h-1). Korekcija pH u svim daljim istraživanjima je vršena sa dodatkom natrijum-hidroksida. U mlečno-kiselim fermentacijama sa različitim početnim koncentracijama redukujućih šećera (2,7; 5,4 i 8,1%) i sa dodatkom različitih koncentracija ekstrakta kvasca (0,5-5,0%), najveći prinos L-(+)-mlečne kiseline i zapreminska produktivnost od 91,29% i 1,69 g/l·h-1, kao i vijabilnost ćelija L. rhamnosus od 9,7·109 CFU/ml ostvareni su u fermentaciji sa početniom koncentracijom redukujućih šećera od 5,4% i dodatkom 5,0% ekstrakta kvasca. Na osnovu ostvarenih rezultata u istraživanjima sa dodatkom džibre i dodacima tokom fermentacije kao i u fermentacijama sa imobilisanim ćelijama je korišćen hidrolizat pivskog tropa sa početnom koncentracijom redukujućih šećera od 5,4%. U mlečno kiseloj fermentaciji sa dodatkom pivskog kvasca najveći prinos L-(+)-mlečne kiseline (89,01%) i zapreminska produktivnost (0,89 g/l·h-1) L-(+)-mlečne kiseline su ostvareni u fermentaciji sa dodatkom 5,0% pivskog kvasca i korekcijom početne koncentracije redukujućih šećera na 5,0%. Na osnovu rezultata utvrđeno je da se može izvršiti delimična ili potpuna zamena ekstrakta kvasca pivskim kvascem uz značajno smanjenje cene podloge za mlečno-kiselu fermentaciju, bez značajnog smanjenja efikasnosti mlečno-kisele fermentacije. U mlečno-kiseloj fermentaciji sa dodatkom džibre i bistre džibre najveć koncetracija, prinos i zapreminska produktivnost L-(+)-mlečne kiseline od 31,03 g/l, 86,15% i 0,93 g/l·h-1, ostvareni su u fermentaciji sa dodatkom 50% bistre džibre. Najviša koncentracija, prinos i zapreminska produktivnost L-(+)-mlečne kiseline ostvareni u dolivnoj fermentaciji sa dodatkom glukoze i bistre džibre tokom mlečno-kisele fermentacije su iznosili su 48,02 g/l, 87,82% i 0,96 g/l·h-1. U fermentacijama sa dodatkom nutritijenata tokom mlečno-kisele fermentacije najveća vrednost koncetracije, prinosa i zapreminske produktivnosti L-(+)-mlečne kiseline od 116,08 g/l, 93,32% i 2,04 g/L·h-1, su ostvarene u fermentaciji sa dodatkom glukoze i ekstrakta kvasca tokom fermentacije. Na osnovu rezultata utvrđeno je da se dolivni postupak fermentacije može koristiti u cilju povećanja efikasnosti mlečno-kisele fermentacije. Izvršena je imobilizacija ćelija L. rhamnosus u kalcijum-alginatu uz izuzetno visoku vijabilnost (1010 CFU/ml). Imobilisane ćelije L. rhamnosus su uspešno korišćene u tri mlečno-kisele fermentacije. Prinos L-(+)-mlečne kiseline i zapreminska produktivnost su u sve tri fermentacije bili izuzetno visoki, pri čemu su najveći prinos L-(+)-mlečne kiseline i zapreminska produktivnost od 95,2% i 1,76 g/l·h-1, ostvareni u drugoj fermentaciji. Upotrebom imobilisanih ćelija L. rhamnosus je osim povećanja prinosa i zapreminske produktivnosti L-(+)-mlečne kiseline skraćena fermentacija za 12 sati u poređenju sa šaržnim fermentacijama.sr
dc.description.abstractBrewers spent grain represents (BSG) about 85% of the total by-products from brewing process and is available at low price during the whole year. Due to its chemical composition BSG has great potential use in biotechnology and production of high-value products. One of very eco-friendly and economical alternative uses of BSG is in production of lactic acid (LA), since in the last few decades the demand for the LA has significantly risen, mostly because of development of biodegradable lactic polymers, which are eco-friendly and nontoxic. Lactic acid is the most important hydrocarboxylic acid with an asymmetrical carbon atom, widely distributed in nature, and it has shown great potential in fields of food, pharmaceutical, textile, leather and chemical industries. The aim of this doctoral thesis was to investigate the application of BSG in lactic acid production. First, the optimization of enzymatic hydrolysis of BSG was conducted, with the goal to achieve high reducing sugar concentrations, as much as possible, that are necessary on LA fermentation. BSG hydrolysis was conducted by usage of commercial enzymes for degradation of starch and cellulose. Effect of pH value, temperature and enzyme dosage on BSG hydrolysis efficiency was investigated. After the best conditions for BSG hydrolysis were determined, the optimized procedure for BSG hydrolysis was used for the production of BSG hydrolysate that will be used in LA fermentations. After optimization of BSG hydrolysis, LA fermentation by two LA producing microorganisms was investigated. The strains investigated were two LA bacteria strains: Lactobacillus fermentum PL-1 and Lactobacillus rhamnosus ATCC 7469. The effect of yeast extract (0.5; 1.0; 2.0; 3.0; 4.0, and 5.0%) addition in BSG hydrolysate, with the correction of pH value during LA fermentation by the addition of calcium-carbonate, on LA fermentation was investigated. Based on the results achieved for L-(+)- and D-(-)-LA ratio the LAB strains that produced more L-(+)-LA was chosen for further research. In further research the effect of pH correction (with addition of NaOH), yeast extract (0.5, 1.0, 2.0, 3.0, 4.0, and 5.0%) addition and reducing sugar concentration (2.7; 5.4 and 8.1%) in BSG hydrolysate on LA fermentation was investigated. Based on the results achieved the best yeast extract and reducing sugars concentrations was determined and used in further analysis or research. Also the possible replacement of expensive yeast extract and glucose with cheap alternatives, like brewer`s spent grain and stillage was investigated. The effect of brewer`s spent grain (0.5; 1.0; 2.0; 3.0; 4.0, and 5.0%), whole stillage (5, 10, 15 i 20%) and thin stillage (5, 10, 15, 20, 30, 40, 50%) addition before fermentation as well as thin stillage addition in fed-batch fermentation in BSG hydrolysate on LA fermentation were investigated. Also fed-batch fermentation procedure (addition of glucose, glucose and yeast extract and wort during fermentation) was investigated. The possible application of cells immobilized in Ca-alginate for LA fermentation of BSG hydrolysate was also investigated. Based on the results it was concluded that BSG can be successfully utilized as a raw material in production of LA, after optimization of hydrolysis and addition of nitrogen source. According to the results of chemical composition before and after optimized hydrolysis 78.6% of total cellulose was hydrolyzed. Addition of calcium-carbonate had positive effect on LA production by L. fermentum i L. rhamnosus. With the addition of calcium-carbonate reducing sugar utilization, LA yield and concentration and cell viability (both L. fermentum i L. rhamnosus) increased. Addition of calcium-carbonate and yeast extract had a positive effect on LA fermentation by L. fermentum and L. rhamnosus. In LA fermentation by L. fermentum the highest LA yield (44%) was achieved with addition of 5.0% of yeast extract and 2.0% of calcium-carbonate. In L. rhamnosus fermentations the highest total LA yield (98%) and L-(+)-LA yield (96%) was reached when 2.0% of yeast extract and 2.0% of calcium-carbonate were added. Based on the results achieved it was concluded that BSG hydrolysate, with the addition of yeast extract, is a good fermentation media for LA fermentation with L. rhamnosus, and it was decided that L. rhamnosus will be used in further research of LA fermentation on BSG hydrolysate. Addition of NaOH instead of calcium-carbonate for the pH correction shortened the fermentation time by 48 h and increased the L-(+)-LA volumetric productivity (by 200%, from 0.21 to 0.63 g/L·h-1). Based on this results pH correction in further experiments was done by addition of NaOH. In LA fermentation with different reducing sugar (2.7, 5.4 and 8.1%) and yeast extract concentrations (0.5-5.0%), the highest L-(+)-LA yield and volumetric productivity of 91.29%, and 1.69 g/L·h-1, respectively, as well as L. rhamnosus cell viability (9.67 log CFU/mL), were achieved with the reducing sugar content of 5.4% and yeast extract content of 5.0%. Based on this results in further experiment with the addition of stillage, in fed-batch fermentation and fermentation with immobilized cell BSG hydrolysate with 5.4% of reducing sugars and 5.0% yeast extract was used. In fermentation with the addition of brewer’s spent yeast the highest L-(+)-LA yield (89.01%) and volumetric productivity (0.89 g/L·h-1) were achieved in the fermentation of BSG hydrolysate with 5.0% of reducing sugar and 5.0% of brewer’s yeast. Based on the results achieved it was concluded that yeast extract can be partial or complete replaced by brewer’s spent yeast with significant decrease of media cost, without the decrease in LA fermentation efficiency. In fermentation with the addition of thin stillage the highest L-(+)-LA concentration, yield, and volumetric productivity of 31.03 g/L, 86.15%, and 0.93 g/L·h-1, respectively, was obtained in fermentation with the addition of 50% of thin stillage. The highest L-(+)-LA concentration, yield, and volumetric productivity achieved in fed-batch fermentation with the addition of glucose and thin stillage during fermentation, were 48,02 g/L, 87,82% i 0,96 g/L·h-1. In fed-batch fermentation the highest L-(+)-LA concentration, yield, and volumetric productivity of 116.08 g/L, 93.32%, and, 2.04 g/L h-1, respectively, were achieved in fermentation with glucose and yeast extract addition during fermentation. The results showerd that fed-batch fermentation could be used to increase L-(+)-LA fermentation efficiency Immobilization of L. rhamnosus cells with high viability (1010 CFU/mL) in Ca-alginate was conducted. Immobilized cells we successfully utilized in three repeated batch fermentation. L-(+)-LA yield and volumetric productivity were very high in all three batch fermentation, with the highest results achieved (95.20% and 1.76 g/L·h-1, respectively) in second fermentation. Application of immobilized L. rhamnosus cells increased L-(+)-LA yield and volumetric productivity and shortened the fermentation time for 12 h in comparison with batch fermentation.en
dc.languagesr (latin script)
dc.publisherУниверзитет у Новом Саду, Технолошки факултетsr
dc.rightsAttribution-NonCommercial-NoDerivs
dc.sourceУниверзитет у Новом Садуsr
dc.subjectPivski trop, mlečno-kisela fermentacija, mlečna kiselina, pivski kvasac, L. rhamnosus, L. fermentumsr
dc.subjectBrewer’s spent grain, lactic acid fermentation, lactic acid, brewer’s spent yeast, L. rhamnosus, L. fermentumen
dc.titlePivski trop – sirovina u mlečno-kiseloj fermentacijisr
dc.title.alternativeBrewer’s spent grain – raw material in lactic acid fermentationen
dc.typeDoktorska disertacijasr


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