fundamental study on the application of lactic acid

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

EBC Symposium Wrocław 2016

Modern brewhouse technologies and wort production

Outline

• Oat

• Barley as a control

• Malting

• Brewing

• Lactic acid bacteria (LAB)

• Exopolysaccharide (EPS)

• Antifungal impact and compounds

20/09/2016 Eric Steffen 2

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 12-15 9-12 10-13

Starch 50-60 60-70 60-70

Fat: 5-8 2-3 2-3

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 maltc

extract [%] dm 62.3a 79.3-81.4 viscosity [mPa*s] 1.43a 1.45-1.90 degree of fermentation [%] EBC 76.6 a <82 protein (conversion factor 5.4/5.5) 10.67a 6.77-10.29 total soluble nitrogen [%] dm 0.60a 0.6-0.8 Kolbach index [%] 30b 35-41 FAN [mg/L] 161b 110-180

α-amylase / AU 95 223

β-amylase / BU 204 1177

Standard deviation a < 2%; b <10%; c: specifications; d: Mäkinen et al. Plant Foods Hum Nutr. 2013 Mar;68(1):90-5.

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Brewing with oat • Oata and husk removed oata

• Grist/water ratio 1:3.6 • rests at 45, 62, 72 °C

• Barley, congress mash

• Shorter lauter times • Higher husk content

• Can benefit perception

• Unique flavour • mouth feeling

20/09/2016 a: Klose et al. (2011), J Inst Brew Volume 117, Issue 3, p.: 411–421 5

0

50

100

150

200

Husk removedoat

Oat Barley

Total sugars in g/l

Mashing regime for oats malt

0

10

20

30

40

50

60

70

80

90

0 20 40 60 80 100 120

mashing time [min]

tem

pera

ture

[˚C

]

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


Effect of LAB-fermentation on beer / beverages • Production of organic acids

• pH reduction

• Nutrients

• Phytate reduction • Increased bioavailability • Bioactive peptides

• Perception

• Organic acids • Flavour compounds • Precursors • Improved texture

Lactic acid

EPS


Current use of LAB in brewing • Grain

• reduction of spoilage organism growth

• Malt

• Improved malting modification • lower malting loss • acid malts

• Brewing

• biological acidification • Sour beer

LAB with antifungal activity

Control LAB


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


Extracellular

Intracellular

gtf123 Glucansucrase gene transcription

Enzyme secreted from cell

Enzyme produced

Sucrose hydrolysed to glucose and fructose

Sequential addition of glucose molecules by the enzyme results in elongation of the glucan polymer – a polysaccharide is made

Fructose is segregated into the medium

The resulting extracellular polysaccharide (EPS) is a chain of linked glucose molecules known as a glucan

Sucrose

Glycansucrase enzyme

Glucose

Fructose

Maltose Glucan

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)

Synthesis of a Glucan Homopolysaccharide

Sucrose present in the surrounding medium

10

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


Growth of LAB in

broth / wort

Centrifugation

Precipitation of dextran with

96% Ethanol

Centrifugation

Dissolve in distilled water

and dialyse

Lyophilize

Supernatant

Dextran

Rheological characterization of dextran in aqueous solution

EPS extraction


+ 10% sucrose

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 MRSsucrose • size of of 5*106 – 4*107 kDa • α-1.6 glucose linkage

• Highest production in MRS 41.6 g/l

• Wort substrates deliver ~10 g/l EPS


0

10

20

30

40

Husk removedOat

Oat Barley MRS

EPS in g/l

Impact of EPS on viscosity • Rheometry of rehydrated EPS

• shear rate of 10 s-1 • Oat / Barley EPS increases viscosity

2.5


1

10

100

Vis

cosi

ty [mPa·s]

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


Mycotoxins in food

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

Antifungal compounds

Cyclic dipeptides

Diacetyl

CO2

Lactic acid

Acetic acid

Phenyllactic acid (PLA)

Propionic acid

Fatty acids

LAB as natural preservatives


Fermentation • Oat, husk removed oat and barley wort

• Fermented with L. reuteri R29; DSM 20016

• Strains have shown antifungal activity • 1% inoculation, 120h fermentation • Changes in sugars, acids monitored

Incubation

Pre inoculum (MRS)

Over night culture

1%

Wort substrate


Fermentation with R29 and DSM 20016 • Oat is a nutritious substrate for LAB

• Decrease in sugars • Increase in organic acids


65

85

105

125

145

0 h 24 h 48 h 72 h 120 h

Total sugars in g/l

1

4

7

10

13

0 h 24 h 48 h 72 h 120 h

Total acids in g/l

• Cell free supernatant (24 -120h fermentation) challenged with fungi for 7 days; two independent triplicates

Microtiter results

• R29 could suppress fungi • in oat and husk

removed oat for 7 days • In barley after 72h

fermentation

• DSM 20016 could suppress

fungi • in husk removed oat for 7

days • after 120 and 72 h

fermentation in oat and barley, respectively

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Barley R29 Oat DSM

Husk removed oat DSM

Barley DSM

Positive controls

Negative controls

24h

48h

72h

120h

24h

48h

72h

120h

Oat R29

Husk removed oat R29

Cell free supernatant

104 Fusarium culmorum spores

7 days Or

• Antifungal activity • No fungal growth

• Overgrown by fungi • No antifungal activity

Well on a 96 well plate:

QuEChERS extraction for antifungal acids

Add 10ml EA with 1% FA

• C18 (150 mg) • MgSO4 (900 mg)


15 antifungal acids analysed

0

10

20

30

40

50

60

24h 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

Hu

sk r

emo

ved

oat

Hu

sk r

emo

ved

oat

aci

dif

ied

Oat

Oat

aci

dif

ied

Bar

ley

Bar

ley

acid

ifie

d

Antifungal compounds in mg/l L. reuteri DSM 20016 L. reuteri R29

Oat Oat, husk removed

Barley Oat Oat, husk removed

Barley

22

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


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 20016 fermentation

• Unique antifungal acid composition found • Fungi suppression evident - 7 days mould free

• R29 after24h (both oats) fermentation (barley 72h) • DSM 20016 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


School of Food and Nutritional Sciences University College Cork College Road, Cork Ireland

Acknowledgements: Prof. Elke Arendt Dr. Emanuele Zannini Dr. Claudia Axel Mr. Alan Lucid Dr. Kieran Lynch And the whole cereal science group at UCC !!!

Thank you for your attention!

This project has received funding from the European Union’s H2020 Research and Innovation Programme under grant agreement n.696300.

JPI - HDHL -healthy diet for healthy life - Longlife - 2016

25 Eric Steffen 20/09/2016

fundamental study on the application of lactic acid

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