LACTIC ACID FERMENTATION OF BREWERS SPENT GRAIN HYDROLYSATE BY LACTOBACILLUS FERMENTUM AND LACTOBACILLUS RHAMNOSUS
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1 LACTIC ACID FERMENTATION OF BREWERS SPENT GRAIN HYDROLYSATE BY LACTOBACILLUS FERMENTUM AND LACTOBACILLUS RHAMNOSUS Jelena Pejin 1*, Ljiljana Mojović 2, Sunčica Kocić- Tanackov 1, Miloš Radosavljević 1, Aleksandra Djukić- Vuković 2 1 Faculty of Technology, University of Novi Sad, Bulevar Cara Lazara 1, Novi Sad, Serbia 2 Faculty of Technology and Metallurgy, University of Belgrade, Karnegijeva 4, Belgrade, Serbia FoodTech Congress, October 28-30, Novi Sad, Serbia
2 PROJECT Ministry of Education, Science and Technological Development of the Republic of Serbia (Project number TR-31017) Title: Increase in bioethanol production efficiency from renewable raw materials with total usage of by-products Leader: Prof. dr Ljiljana Mojović, Faculty of Technology and Metallurgy, University of Belgrade
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6 PROJECT Ministry of Education, Science and Technological Development of the Republic of Serbia (Project number TR-31017) Title: Production of lactic acid and probiotics on waste products of food and agro-industry Leader: Prof. dr Ljiljana Mojović, Faculty of Technology and Metallurgy, University of Belgrade 2011-
7 Total usage of triticale Triticale stillage obtained after bioethanol production was used for lactic acid production Liquid triticale stillage was a good raw material for lactic acid fermentation.
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10 Beer is the most commonly consumed alcoholic beverage in the world
11 Beer is a malt beverage produced by an alcoholic fermentation of the aqueous of malted barley with hops. Brewing is therefore a multistage process involving biological conversion of raw materials to final product.
12 Beer production scheme
13 BREWERS SPENT GRAIN
14 Brewers spent grain (BSG) is the major by-product of the brewing industry, representing around 85% of the total by-products generated. Per 100 L of beer produced 20 kg of brewers spent grain are obtained. The chemical composition of brewers spent grain varies according: to barley variety, harvest time, malting and mashing conditions, and the quality and type of adjuncts used in the brewing process.
15 Brewers spent grain is a lignocellulosic material rich in protein and fibre, which account for around 20 and 70% of its composition, respectively. Component (% dry matter) Bogar et al. (2002) Mussatto and Roberto (2005) Serena and Knudsen (2007) Dehnavi (2009) Cellulose Hemicellulose Lignin Proteins Ash Carbohydrates Lipids Starch
16 The use of brewer s spent grain is still limited, being basically used as animal feed and in human nutrition.
17 Its possible applications are as a raw material in: biotechnology, energy production, charcoal production, paper manufacture, Or as a brick component, and adsorbent.
18 Yeast carrier in beer fermentation Microorganisms Enzymes Biogas Brewers spent grain Phenolic acids Bioethanol Lactic acid Xylitol and pullulan
19 Lactic acid
20 Currently, there is an increased demand for lactic acid as a raw material for the production of biopolymer poly-lactic acid (PLA) which is a promising biodegradable, biocompatible, and environmentally friendly alternative to plastics derived from petrochemicals. Food and food-related applications account for approximately 85% of the demand for lactic acid. The demand for lactic acid has been estimated to grow yearly at 5 8%.
21 The annual world market for lactic acid production was expected to reach 367,300 metric tons by the year There are two optical isomers of lactic acid, L- (+)-lactic acid and D-(-)-lactic acid. Lactic acid can be manufactured either by chemical synthesis or by microbial fermentations.
22 Presently, almost all lactic acid produced worldwide comes from the fermentative production route. A desired isomer of lactic acid can be produced via fermentation using selected lactic acid-producing strains. Besides this, microbial lactic acid fermentation offers an advantage in terms of the utilization of renewable carbohydrate biomass, low production temperature and energy consumption.
23 Most lactic acid bacteria require a wide range of growth factors including amino acids, vitamins, fatty acids, purines, and pyrimidines for their growth and biological activity. Thus, the substrate composition and nutritional requirements of the strain considerably affect the overall performance of the fermentation.
24 Brewers spent grain During the fermentation, determination of: Lactic acid concentration (L-/D-lactic acid assay, Megazyme, Wicklow, Ireland) Reducing sugar content (Miller, G. L. (1959). Anal. Chem., 31, Cells viability (pour plate technique, MRS agar, 30 C or 37 C) ph value Brewers spent grain hydrolysis ph 5.5 and 5.0 Termamyl SC (1 hour at 90 C), SAN Super 240 L (1 hour at 55 C), Celluclast 1.5 L (10 hours at 45 C) at 180 rpm Centrifugation Sterile liquid hydrolysate fermentation media CaCO 3 or NaOH Reducing sugars content Yeast content Fermentation L. fermentum PL-1 at 30 C L. rhamnosus ATCC 7469 at 37 C 150 rpm Lactic acid
25 BREWERS SPENT GRAIN HYDROLYSIS OPTIMIZATION Brewers spent grain obtained in a lager beer production was dried at 40ºC for 12 hours. Brewers spent grain hydrolysis was carried out under optimal conditions using the following enzymes: 1. Termamyl SC - α-amylase, 2. SAN Super 240 L glucoamylase, and 3. Celluclast 1.5 L cellulase (Novozymes, Denmark).
26 Different Temperatures: Termamyl SC 80, 85 or 90ºC (1 hour) San Super 240 L 50, 55 or 60ºC (1 hour) Celluclast 1.5 L 40, 45 or 50ºC (10 hours) ph values: 5.0, 5.5 and 6.0 for each enzyme Amounts: Termamyl SC 0.1, 0.2, 0.3 ml pre 50g San Super 240 L 0.1, 0.2, 0.3 ml pre 50g Celluclast 1.5 L 3.0, 4.0, 5.0 or 6.0 ml per 50g
27 Reducing sugars content, g/l 30 ph 5.5 Termamyl SC and San Super 240 L, ph 5.0 Celluclast 1.5 L ph mL TSC; 0.3mL San Super; ph mL Celluclast ph mL TSC; 0.3mL San Super; ph mL Celluclast ph 5.5 0,3mL TSC; 0.3mL San Super; ph mL Celluclast ph mL TSC; 0.3mL San Super; ph mL Celluclast Time, hours
28 Lactic Lactic acid acid content content, (g/l) g/l L-(+)-lactic acid increase % Without calcium-carbonate 3.0% 0.5% of addition L-(+)-lactic acid acid Without calcium-carbonate 3.0% 0.5% of addition D-(-)-lactic acid acid With calcium-carbonate 4.0% 1.0% of addition L-(+)-lactic acid acid 1.0% With calcium-carbonate 4.0% of addition D-(-)-lactic D-(-)-lactic acid acid 5.0% 2.0% of L-(+)-lactic acid % 2.0% of 48 D-(-)-lactic acid Time, hours Time, hours Lactic acid content in Lactobacillus fermentum PL-1 fermentations
29 Viability, log CFU/mL % increase % increase % of % of 2.0% of % of % Without of calcium-carbonate addition 5.0% With of calcium-carbonate addition Time, hours Time, hours Lactobacillus fermentum PL-1 cells viability during fermentations
30 Lactic Lactic acid acid content, g/l g/l g/l L-(+)-lactic acid content increase % L-(+)-lactic content increase 1248% % of L-(+)-lactic acid 0.5% Without calcium-carbonate 3.0% of of D-(-)-lactic addition L-(+)-lactic acid D-(-)-lactic acid 1.0% acid With calcium-carbonate 3.0% of of L-(+)-lactic addition D-(-)-lactic acid L-(+)-lactic acid 1.0% of D-(-)-lactic acid With calcium-carbonate 4.0% of 2.0% of addition L-(+)-lactic L-(+)-lactic D-(-)-lactic acid 4.0% of D-(-)-lactic acid acid 2.0% 5.0% of of D-(-)-lactic L-(+)-lactic acid acid Without calcium-carbonate addition L-(+)-lactic acid Time, hours Lactic acid content in Lactobacillus rhamnosus ATCC 7469 fermentations
31 Viability, log log CFU/mL % increase % increase Without calcium-carbonate addition With calcium-carbonate addition Time, hours hours 0.5% of 1.0% of 2.0% of 3.0% of 4.0% of 5.0% of Lactobacillus rhamnosus ATCC 7469 cells viability during fermentations
32 Total lactic acid yield, % Lactobacillus fermentum PL-1 Lactobacillus rhamnosus ATCC Without CaCO₃ With CaCO₃ 0.5% of 1.0% of 2.0% of 3.0% of 4.0% of 5.0% of Lactic acid fermentation
33 Brewers spent grain During the fermentation, determination of: Lactic acid concentration (L-/D-lactic acid assay, Megazyme, Wicklow, Ireland) Reducing sugar content (Miller, G. L. (1959). Anal. Chem., 31, Cells viability (pour plate technique, MRS agar, 30 C or 37 C) ph value Brewers spent grain hydrolysis ph 5.5 and 5.0 Termamyl SC (1 hour at 90 C), SAN Super 240 L (1 hour at 55 C), Celluclast 1.5 L (10 hours at 45 C) at 180 rpm Centrifugation Sterile liquid hydrolysate fermentation media NaOH Reducing sugars content (2.7, 5.4, and 8.1%) Yeast content ( %) Fermentation L. rhamnosus ATCC 7469 at 37 C 150 rpm Lactic acid
34 L-(+)-lactic acid content, g/l % of reducing sugars % increase Time, hours Without addition 0.5% of 1.0% of 2.0% of 3.0% of 4.0% of 5.0% of
35 L-(+)-lactic acid content, g/l Without addition 0.5% of 1.0% of 2.0% of 3.0% of 4.0% of 5.0% of 5.4% of reducing sugars % increase Time, hours
36 L-(+)-lactic acid content, g/l % of reducing sugars % increase Time, hours Without addition 0.5% of 1.0% of 2.0% of 3.0% of 4.0% of 5.0% of
37 Viability, log CFU/mL % of reducing sugars % increase Without addition 0.5% of 1.0% of 2.0% of 3.0% of 4.0% of 5.0% of Time, hours
38 Viability, log CFU/mL % of reducing sugars % increase Without addition 0.5% of 1.0% of 2.0% of 3.0% of 4.0% of 5.0% of Time, hours
39 Viability, CFU/mL % of reducing sugars % increase Without addition 0.5% of 1.0% of 2.0% of 3.0% of 4.0% of 5.0% of Time, hours
40 L-(+)-lactic acid yield, % % increase Without addition 0.5% of 1.0% of 2.0% of 3.0% of 4.0% of 5.0% of Lactic acid fermentation
41 L-(+)-lactic acid yield, % % increase Without addition 0.5% of 1.0% of 2.0% of 3.0% of 4.0% of 5.0% of Lactic acid fermentation
42 L-(+)-lactic acid yield, % % increase Without addition 0.5% of 1.0% of 2.0% of 3.0% of Lactic acid fermentation 4.0% of 5.0% of
43 Volumetric productivity (g/l h) Time, hours Without addition 0.5% of 1.0% of 2.0% of 3.0% of 4.0% of 5.0% of
44 Volumetric productivity (g/l h) Time, hours Without addition 0.5% of 1.0% of 2.0% of 3.0% of 4.0% of 5.0% of
45 Volumetric productivity (g/l h) Time, hours Without addition 0.5% of 1.0% of 2.0% of 3.0% of 4.0% of 5.0% of
46 Brewers spent grain During the fermentation, determination of: Lactic acid concentration (L-/D-lactic acid assay, Megazyme, Wicklow, Ireland) Reducing sugar content (Miller, G. L. (1959). Anal. Chem., 31, Cells viability (pour plate technique, MRS agar, 30 C or 37 C) ph value Brewers spent grain hydrolysis ph 5.5 and 5.0 Termamyl SC (1 hour at 90 C), SAN Super 240 L (1 hour at 55 C), Celluclast 1.5 L (10 hours at 45 C) at 180 rpm Centrifugation Sterile liquid hydrolysate fermentation media NaOH Brewers ( %) Reducing sugars content Fermentation L. rhamnosus ATCC 7469 at 37 C 150 rpm Lactic acid
47 The possibilities of using spent brewers (Spent Brewer s Yeast and Beta-Glucans Isolated From Them as Diet Components Modifying Blood Lipid Metabolism Disturbed by an Atherogenic Diet, B. Waszkiewicz- Robak, 2013)
48 L-(+)-lactic acid content, g/l % increase Without brewers' addition 0.5% of brewers' 1.0% of brewers' 2.0% of brewers' 3.0% of brewers' 4.0% of brewers' 5.0% of brewers' Time, hours
49 L-(+)-lactic acid content, g/l 30 5% of reducing sugars % increase Time, hours Without brewers' addition 0.5% of brewers' 1.0% of brewers' 2.0% of brewers' 3.0% of brewers' 4.0% of brewers' 5.0% of brewers'
50 Viability, log CFU/mL % increase Without brewers' addition 0.5% of brewers' 1.0% of brewers' 2.0% of brewers' 3.0% of brewers' 4.0% of brewers' 5.0% of brewers' Time, hours
51 Viability, log CFU/mL 9.8 5% of reducing sugars % increase Time, hours Without brewers' addition 0.5% of brewers' 1.0% of brewers' 2.0% of brewers' 3.0% of brewers' 4.0% of brewers' 5.0% of brewers'
52 L-(+)-lactic acid yield, % % increase Without brewers' addition 0.5% of brewers' 1.0% of brewers' 2.0% of brewers' 3.0% of brewers' Lactic acid fermentation 4.0% of brewers' 5.0% of brewers'
53 L-(+)-lactic acid yield, % % increase Without brewers' addition 0.5% of brewers' 1.0% of brewers' 2.0% of brewers' 3.0% of brewers' Lactic acid fermentation 4.0% of brewers' 5.0% of brewers'
54 Time, hours Without brewers addition 0.5% of brewers Volumetric productivity (g/l h) 1.0% of brewers 2.0% of brewers 3.0% of brewers 4.0% of brewers 5.0% of brewers Time, hours Without brewers addition 0.5% of brewers Volumetric productivity (g/l h) 1.0% of brewers 2.0% of brewers 3.0% of brewers 4.0% of brewers 5.0% of brewers
55 Brewers spent grain Brewers spent grain hydrolysis ph 5.5 and 5.0 Termamyl SC (1 hour at 90 C), SAN Super 240 L (1 hour at 55 C), Celluclast 1.5 L (10 hours at 45 C) at 180 rpm During the fermentation, determination of: Lactic acid concentration (L-/D-lactic acid assay, Megazyme, Wicklow, Ireland) Reducing sugar content (Miller, G. L. (1959). Anal. Chem., 31, Cells viability (pour plate technique, MRS agar, 30 C or 37 C) ph value Centrifugation Sterile liquid hydrolysate fermentation media NaOH Glucose (after 12, 24, 36, and 48 hours) to the initial content Glucose and (after 12, 24, 36, and 48 hours) to the initial content High gravity wort (10 ml every 4 hours) Fermentation L. rhamnosus ATCC 7469 at 37 C 150 rpm Lactic acid
56 L-(+)-lactic acid content, g/l Without glucose and addition during fermentation With glucose addition With glucose and addition With wort addition % increase % increase Time, hours
57 Viability, log CFU/mL Without glucose and addition during fermentation With glucose addition With glucose and addition With wort addition Time, hours
58 L-(+)-lactic acid yield, % Without glucose and addition during fermentation With glucose addition With glucose and addition Lactic acid fermentation With wort addition
59 Time, hours Without glucose and addition during fermentation Volumetric productivity (g/l h) With glucose addition With glucose and addition With wort addition
60 Colleagues: Ljiljana Mojović Sunčica Kocić-Tanackov Miloš Radosavljević Aleksandra Djukić-Vuković Ministry of Education, Science and Technological Development of the Republic of Serbia (Project number TR-31017) Carlsberg Serbia Novozymes, Denmark
61 THANK YOU KINDLY!
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