Fundamental study on the application of lactic acid bacteria in oat wort based beverages

Transcription

1 EBC Symposium Wrocław 2016 Modern brewhouse technologies and wort production Fundamental study on the application of lactic acid bacteria in oat wort based beverages Eric Steffen, Kieran Lynch, Alan Lucid, Claudia Axel, Elke K. Arendt School of Food and Nutritional Sciences University College Cork

2 Outline Oat Barley as a control Malting Brewing Lactic acid bacteria (LAB) Exopolysaccharide (EPS) Antifungal impact and compounds 20/09/2016 Eric Steffen 2

3 Why Oat? Economy Lower prices Frost resistant Low sensitivity to diseases and rain Global production (Russia, EU, USA, Canada, Australia) Nutrition Highest protein amongst cereals Fatty acids Non-digestible fibre β-glucan Challenge: Utilize the properties and improve value by fermentation with LAB [%] Oats Barley Wheat Protein Starch Fat: /09/2016 Eric Steffen 3

4 Oat malting Oat (Avena sativa) d steeped, germinated at 15 C for 5 days kilned in three stages (35 C, 50 C and 60 C) Barley (Hordeum vulgare) Pilsner malt Malt analysis: Oats malt Barley malt c extract [%] dm 62.3 a viscosity [mpa*s] 1.43 a degree of fermentation [%] EBC 76.6 a <82 protein (conversion factor 5.4/5.5) a total soluble nitrogen [%] dm 0.60 a Kolbach index [%] 30 b FAN [mg/l] 161 b α-amylase / AU β-amylase / BU /09/2016 Standard deviation a < 2%; b <10%; c: specifications; 4 d: Mäkinen et al. Plant Foods Hum Nutr Mar;68(1):90-5.

5 temperature [ C] Brewing with oat Oat a and husk removed oat a Grist/water ratio 1:3.6 rests at 45, 62, 72 C Barley, congress mash Mashing regime for oats malt mashing time [min] Shorter lauter times Higher husk content Can benefit perception Unique flavour mouth feeling Total sugars in g/l 0 Husk removed oat Oat Barley 20/09/2016 a: Klose et al. (2011), J Inst Brew Volume 117, Issue 3, p.:

6 What are lactic acid bacteria (LAB)? Lactobacillus, Leuconostoc, Pediococcus, and Streptococcus form the core of the group Gram-positive, non spore-forming, rod or cocci produce lactic acid as the major end-product Consumer demand for natural, healthy products Use of LAB to extend shelf life and quality Beneficial effects on human health Several LAB have the GRAS (US) and (QPS) (EFSA) status 20/09/2016 Eric Steffen 6

7 Effect of LAB-fermentation on beer / beverages Production of organic acids ph reduction Nutrients Phytate reduction Increased bioavailability Bioactive peptides Lactic acid Perception Organic acids Flavour compounds Precursors Improved texture EPS 20/09/2016 Eric Steffen 7

8 Current use of LAB in brewing Grain reduction of spoilage organism growth Malt Improved malting modification lower malting loss acid malts Control LAB LAB with antifungal activity Brewing biological acidification Sour beer 20/09/2016 Eric Steffen 8

9 What is EPS? extracellularly secreted microbial polysaccharides Increase in viscosity Amount or structure is strain and substrate dependant Homopolysaccharides Long chains of monosaccharides Extracellular synthesis Up to 50 g/l Heteropolysaccharides Intracellular synthesis from sugar nucleotides Low yields < 2g/l Mutan, levan Levan 20/09/2016 Eric Steffen 9

10 Sucrose Glycansucrase enzyme Glucose Fructose Maltose Extracellular Intracellular Enzyme produced gtf123 Glucansucrase gene transcription If maltose, for example, is present in the medium it acts as an acceptor sugar leading to shorter molecules an oligosaccharide is formed Gluco-oligosaccharide (GOS) Sucrose present in the surrounding medium Synthesis of a Glucan Homopolysaccharide Glucan Sequential addition of Sucrose hydrolysed to glucose molecules by glucose and fructose the enzyme results in elongation of the glucan polymer a polysaccharide The resulting is made Enzyme secreted extracellular from cell polysaccharide (EPS) is a chain of linked glucose Fructose is segregated molecules into known the medium as a glucan 10

11 Effects of EPS on beverages Increase stability Top fermented beer Non-alcoholic beverages Emulsions Particle suspension Increase of viscosity Gelling, thickening properties From juice-like to honey-like 20/09/2016 Eric Steffen 11

12 EPS extraction Growth of LAB in broth / wort + 10% sucrose Centrifugation Supernatant Centrifugation Dextran Dissolve in distilled water and dialyse Precipitation of dextran with 96% Ethanol Lyophilize Rheological characterization of dextran in aqueous solution 20/09/2016 Eric Steffen 12

13 Fermentation with Weisella cibaria MG1 Worts produce comparable amounts of EPS Viscosity in fermented beverage is increased MRS control had gel like viscosity EPS from MRS sucrose size of of 5*10 6 4*10 7 kda α-1.6 glucose linkage EPS in g/l Highest production in MRS 41.6 g/l 10 0 Husk removed Oat Oat Barley MRS Wort substrates deliver ~10 g/l EPS 20/09/2016 Eric Steffen 13

14 Viscosity [mpa s] Impact of EPS on viscosity Rheometry of rehydrated EPS shear rate of 10 s -1 Oat / Barley EPS increases viscosity /09/2016 Eric Steffen 14

15 Are fungi a problem? Aflatoxin (Aspergillus / Penicillium sp) Toxic to liver, kidneys, nervous system; carcinogenic Trichotecenes A/B (Fusarium sp) Negative effects on gastro-intestinal tract, DNA / protein synthesis; toxic Fumonisins (Fusarium sp) Toxic to nervous system; possible carcinogenic AAL Toxins (Alternaria sp) Fungi are a cause for economic losses and product safety 20/09/2016 Eric Steffen 15

16 Mycotoxins in food 20/09/2016 RASFF - Rapid Alert System for Food and Feed 16

17 LAB as natural preservatives Fatty acids CO2 Phenyllactic acid (PLA) Diacetyl Antifungal compounds Acetic acid Cyclic dipeptides Propionic acid Lactic acid 20/09/2016 Eric Steffen 17

18 Fermentation Oat, husk removed oat and barley wort Fermented with L. reuteri R29; DSM Strains have shown antifungal activity 1% inoculation, 120h fermentation Changes in sugars, acids monitored Over night culture 1% Incubation Pre inoculum (MRS) Wort substrate 20/09/2016 Eric Steffen 18

19 Fermentation with R29 and DSM Oat is a nutritious substrate for LAB Decrease in sugars Increase in organic acids 13 Total acids in g/l 145 Total sugars in g/l h 24 h 48 h 72 h 120 h 65 0 h 24 h 48 h 72 h 120 h 20/09/2016 Eric Steffen 19

20 24h 48h 72h 120h 24h 48h 72h 120h Microtiter results Cell free supernatant (24-120h fermentation) challenged with fungi for 7 days; two independent triplicates Husk removed oat R29 Cell free supernatant Well on a 96 well plate: 10 4 Fusarium culmorum spores Oat R29 Barley R29 Husk removed oat DSM Oat DSM Barley DSM 7 days Positive controls Negative controls R29 could suppress fungi in oat and husk Antifungal removed activity oat for 7 days No fungal In barley growth after 72h fermentation DSM could suppress Or fungi in husk removed oat for 7 days after 120 and 72 h fermentation in oat and Overgrown barley, by fungi respectively No antifungal activity 20/09/

21 QuEChERS extraction for antifungal acids Add 10ml EA with 1% FA 15 antifungal acids analysed C18 (150 mg) MgSO4 (900 mg) 20/09/2016 Eric Steffen 21

22 Husk removed oat Husk removed oat acidified Oat Oat acidified Barley Barley acidified Antifungal compounds in mg/l L. reuteri R29 L. reuteri DSM Oat, husk removed Oat Barley Oat, husk removed Oat Barley h 48h 72h 120h 24h 48h 72h 120h 24h 48h 72h 120h 24h 48h 72h 120h 24h 48h 72h 120h 24h 48h 72h 120h A - Cathecol B - Hydroxyphenyllactic acid C - 4-Hydroxybenzoic acid D - Hydrocaffeic acid E - Vanillic acid G - Phloretic acid H - Phenyllactic acid I - Coumaric acid J - Hydroferulic acid K - Benzoic acid L - Ferulic acid M - Salicylic acid O - Methylcinnamic acid 22

23 Antifungal components conclusion Substrate and strain specific composition of antifungal acids were found Oat, husk removed: Phenyllactic Hydroferulic (R29) Benzoic Catechol (DSM) Hydroxyphenyllactic (DSM) Oats Phenyllactic Hydroferulic (R29) Benzoic Coumaric Methylcinnamic Catechol (DSM) Barley Phenyllactic Hydroxyphenyllactic Hydrocaffeic Catechol Methylcinnamic 20/09/2016 Eric Steffen 23

24 Overall conclusion W. cibaria MG1 fermentation ~10 g/l EPS in both oats and barley Increased viscosity; comparable to milk Improved organoleptic perception L. reuteri R29 and DSM fermentation Unique antifungal acid composition found Fungi suppression evident - 7 days mould free R29 after24h (both oats) fermentation (barley 72h) DSM after 24h for husk removed oat and 120h fermentation for whole oat (barley 72h) Oat and husk removed oat is a suitable substitute to barley for LAB fermentation 20/09/2016 Eric Steffen 24

25 School of Food and Nutritional Sciences University College Cork College Road, Cork Ireland Thank you for your attention! Acknowledgements: Prof. Elke Arendt Dr. Emanuele Zannini Dr. Claudia Axel Mr. Alan Lucid Dr. Kieran Lynch And the whole cereal science group at UCC!!! This project has received funding from the European Union s H2020 Research and Innovation Programme under grant agreement n JPI - HDHL -healthy diet for healthy life - Longlife /09/2016 Eric Steffen 25