Food Fermentation: Microbes, Metabolism, and Methods

What is fermentation?

conversion of organic compounds--primarily sugars--into simpler substances, often in the absence of oxygen.

name some substrates

Plants: fruit, vegetables, legumes

meat, dairy, eggs

name some methods of fermentation

liquid fermentation - wet brine

solid state fermentation - dry cure

submerged fermentation - inoculation

name some of the changed products of fermentation

proteins

acids

sugars / starches

lignin / fibers / connective tissues

what is an example of fermentation happening in your body?

in your muscles: ATP is used for energy in cells, and it is stored in muscle and it only lasts a few seconds, therefore you make new ATP through lactic acid fermentation

Explain the Lactic Acid cycle

- also known as the CORI cycle

- a metabolic pathway connecting Skeletal Muscle tissues and the Liver.

- It describes how lactate produced under low-oxygen conditions is transported, converted back into glucose, and returned to the body

Glucose is used to create energy via glycolysis. What happens when there is a lack of O2

without much O2 your glucose gets shunted down the lactic acid pathway, rather than the respiration pathway (not as efficient)

what are the microorganisms in your gut?

- lactobacillus

- bifidobacteria

- streptococcus thermophilus

- saccharomyces boulardii

What are the two types of fermentation and what are the key organisms for this?

- lactic acid fermentation: produces lactic acid molecules from pyruvate

- alcoholic fermentation: produces ethanol and CO2 from pyruvate

- both of the above use the pyruvate from glycolysis

- ALSO anaerobic fermentations!

- key orgs are bacteria and yeast

What are firmicutes

- prokaryotes

- lactobacillales

- bacillus

- brevibacterium

what are proteobacteria

- prokaryotes

- vinegar agents

- spoil aclohol

what are actinobacteria

- prokaryotes

- bifidobacterium

- kocuria and staphylococcus/micrococcus

- propionibacterium

Lactic acid bacteria!

- lactobacillales and lactic acid bacteria (LABs)

- bacillus (natto)

- brevibacterium (aerobic, shaped weird, certain cheeses ripening and appearance)

- gram positive

- fermentative

- catalase negative

- facultative anaerobic

- non-spore forming

- non-motile

- acid tolerant

gram negative vs. gram positive

gram negative: tick peptidoglycan layer absorbs surrounding materials, even toxins. Easier to kill, develops resistance slower

gram positive: thin peptidoglycan layer covered by multiple thin layers of membrane which eject toxins. Harder to kill, quick to develop resistance

There are different types of LABs and

some are homo and some are hetero. What does this mean?

if homo, it means they mostly produce lactic acid, if hetero, there are multiple end products

what lactobacillales make milk?

- lactococcus lactis

what lactobacillales make yogurt and cheese?

streptococcus thermophilus

What are the proteobacteria in question?

acetobacter and gluconobacter

- gram negative

- obligate aerobes = respire (spoil wine and beer)

- acetobacter aceti - manufacture vinegar

What are the actinobacteria in question?

- bifidobacterium

- kocuria and staphylococcus/micrococcus

- propionbacterium

what is bifidobacterium

have fermentative metabolism but are not natural in any fermented products, but have great health benefits in human gut = we add them to starter cultures on an industrial level now to keep you healthy

what is kocuria and staphylococcus/micrococcus

aerobic, used to enhance flavor, aroma, and appearance of fermented sausages ( do not produce the acid though because not fermentative)

what is propionbacterium

acne and eye maker in swiss chease, not primary fermenting agent in swiss cheese but responsible for the CO2 that forms the holes and propionic acid

Fungi are eukaryotes, and what in this class are we looking at in this kingdom

molds and yeasts

what is ascomycota

Yeasts: saccharomyces, kluyveromyces, zygosaccharomyces

- single celled

- sexual spore producing OR non-sporulating

- budding asexual reproduction

- beer, wine, spirits, and bread

what is zygomycota

molds: aspergillus, penicillium, rhizopus

- multicellular

- complex structure

- asexual and sexual spore producing

- tempeh, soy sauce, soy paste, rice wine, blue mold based cheeses, spoilage

saccharomyces cerevisiae

ALES, BEER, LAGER

explain sugar metabolism

- transforming sugars into energy

- you do respiration when oxygen is around and fermentation when oxygen is not around

homofermentation

- results in a single end product (dry ferments)

- LAB that produce only lactic acid

- EMP (Embden-Meyerhod-Parnas) pathway (glucose into lactic acid)

heterofermentation

- multiple end products

- lactic acid, ethanol, and maybe others

- which product depends on situation

- phosphoketolose pathway

pyruvate importance

- precursor to many things and a key intermediate of lactic acid fermentation (precursor for flavors)

what makes fermentation in yeast different

- pyruvate is decarboxylated to acetaldehyde (which goes on to EtOH)

What are the systems in LABs vs. yeasts for sugar import

- PEP-dependent transferase system

- ABC transporters

- Ion gradients

- yeast has multiple mechanisms

protein metabolism

- important in LAB in yogurt

- LAB cannot use inorganic nitrogen

- casen, a milk protein, is critical for this

citrate formation

- common in plants

- broken down into pyruvate

propionic acid fermentation

- propionibacterium freudenreichii

- eats lactic acid, makes propionic acid and CO2

zymology

the study of fermentation

What is 16S

the 16S ribosomal RNA bacteria species gene and it is like a bacterial fingerprint

- it has variable and conserved regions

- well characterized across many taxa

challenges of 16S

- some variable regions not well represented in databases

- some primer binding sites "miss" particualr taxa

- some variable regions do not resolve species well

eukaryotic 18S rRNA and Internal Transcribed Spacers (ITS)

- ITS1 and ITS2 are hypervariable

- mycobiome (fungal cells) is orders of magnitude smaller than the bacterial microbiome

- does not resolve some species well

- PCR amplification can be difficult

- databases not well characterized

what is OTU

operational taxonomic unit

what is ASV

amplicon sequence variant

species abundance

the total number of organisms in a sample

species richness

the total number of different species in a sample

species evenness

how evenly distributed the species are in a sample

diversity

accounts for both richness and evenness

why ferment?

- nutrition: benefits

- function: transform ingredients into new things

- organoleptic: raw ingredients now taste different

- uniqueness: only way to make these products, cannot make beer without it, parmesan is only parmesan if made a certain way

- economics: milk into cheese and the margin can be insane

What was a key turning point in human evolution for fermentation

the alcohol dehydrogenase enzyme

When was fermentation thought to have arrived

neolithic period

- also a huge part of culture (religion)

what happened in 11000 BCE

as glaciers retrat in the North, wild grains begin to grow in the Near East

what happened in 54 BCE

julius caesar reports cheddar cheese being made in britain

what happened in 1383 AD

a brewery opens in Bavaria

what happened in 1630 AD

soy sauce introduced in Japan by a company that will become Kikkoman

what happened in 1698 AD

the French Benedictine monk, Dom Pierre Perignon developed techniques for retaining CO2 in wine, leading to champagne

what happened in 1743?

the worlds now largest Champagne producer began operations in france

what happened in 1775

captain cook, by feeding saurkraut to his crewman, is awarded a medal by the royal society for conquering scurvy

what happened in 1796

Nicole-Barbe Cliquot invented the remuage technique, used to remove yeast sediment from champagne

what happened in 1820

Frederick Accum, fled to escape industry wrath for a publication that revealed the identities of producers of wine, beer, bread, vinegar, cheese, pickles, and other products that were deliberately adulterated.

What happened in 1850

sourdough bread became a staple in California

1675- Leeuwenhowek discovered what

animalcules

who determined that fermentation is a living process?

louis pasteur

how does pasteurization work?

Pasteurization works by heating up the milk to a high temperature for a certain length of time and then rapidly cooling it. this kills the enzymes and then they are unable to function in the process of helping bacteria grow

what happened in 1857

Louis Pasteur shows that bacteria are responsible for the lactic acid fermentation in milk

what happened in 1880

Heinz Co. introduces White Vinegar and Apple Cider Vinegar

what happened in 1950

bacteriophage against lactic acid bacteria identified by New Zealand researchers

what happened in 1974

miller brewing co. introduced miller lite

what happened in 2006

comparative genome analysis of eleven lactic acid and related bacteria is published

How many fermented beverages are listed on wikipedia

85

What is AAB

acetic acid bacteria

What is SCOBY

Symbiotic Culture Of Bacteria and Yeast

- Noma has examples of training their SCOBYs to carry out ferments in unexpected environments that are not super favorable

How does kefir come to be

kefir grains - tibicos (water)

SCOBY

(water vs dairy kefir)

Dairy kefir

- SCOBY composition is distince from water kefir grains

- kefir can no more be made by the right mix of chemicals or microbes that can oak trees or elephants

- symbiotic entity

- we have not been able to produce it by just putting pieces together

What are spoilers of SCOBY?

- Kham yeast, most common spoiler of SCOBY beverage ferments

- Aspergillus (or penicillium), second common spoiler of SCOBY beverage ferments

what is sanger sequencing

Sanger sequencing is a method for determining the nucleotide sequence of DNA using chain-terminating inhibitors.

What is Oxford Nanopore sequencing?

a third-generation, single-molecule method used to decode DNA and RNA by measuring changes in electrical current as strands pass through microscopic pores

what is Chhü

Yeast + bacteria cultures dominated by mold (chinese word)

- no english equivalent word

- enzymes break off starch and sugar of substrate, mold spores germinate, yeast ferment the sugar to alcohol

- mold needs oxygen to grow = "fermentation"

- aspergillus orzae: treasure trove of enzymes produced, koji

- rhizopus oligosporus: treasure trove of enzymes produced, tempeh

what does not need a starter and what does?

- fruits and veggies do not need it

need:

- dairy to yogurt and cheese

- sourdough starter

- tempeh best produced from starter

pickling vs. fermentation

pickling = anything preserved by acidity

- modern pickles not fermented at all - highly acidic vinegar heated to sterilize vegetable = kill microorgs., not cultivate them

1. chop

2. salt (dry or brine)

3. submerge

4. enjoy

what does salt do for lacto fermenting fruits and veggies

- pulls water out of veggies via osmosis

- harden plant cell pectins = crispier

- slows down pectin-digesting enzymes = crispier

- creates selective environment - only salt tolerant LAB can live

- slows fermentation and development of surface moles = more potential for preservation

what uses these salts/brine

krauts and kimchi

what is yogurt fermented with

lactic acid bacteria

what is the enemy of the yogurt culture

bacteriophage

- affects all LAB

- not known until 1915-1917

- 50-200 progeny per infection

- can suppress a culture within a few hours

- routine screening of cultures required

what are exopolysaccharides

- prebiotics

- substances that promote the growth or activity of beneficial microorganisms

- non-digestible, predominantly bacterially digestible

- immunostimulatory activity, anti-tumoral activity, reduced blood cholesteral levels

probiotics

- probiotics are live microorganisms that, confer a health benefit on the host

- most commercial bacteria in yogurt do not last long

what is gluten?

gliadin + glutenin + water = disulfide bond formation from mixing with water = gluten "mesh" structure

what is bread yeast

- saccharomyces cerevisiae

- what makes bread ride through the production of CO2 (good yeast strains grow easily in flour and produce a lot of gas)

- sensitive to environment

- baker strains have lower genetic diversity and a higher number of chromosomes than other S. cerevisiae lineages tied to human influence, such as wine, sake and beer yeasts

- bakers yeasts were brewers yeasts until 1790s

- now, bakers yeast is mass produced

kneading is necessary, why?

yeast shapes gluten well. it aligns the gluten in the dough so that it is depolymerized

what makes sourdoughs special

- flavor

- texture

- yeast

- LABs (some AABs)

- bread predates Egyptians, but clear evidence for sourdoughs

what is starter vs. levain

the starter is the savings account (mother) and the levain is the checking account (freshly fed starter/mother)

alpha diversity

mean species diversity in a habitat at a local scale (richness vs. evenness)

gamma diversity

total species diversity in a landscape

beta diversity

the ratio between regional and local species diversity (gamma/alpha)

- species turnover

what is Simpson 1-D

- useful in comparing diversity across different habitats/ecosystems

- probability of similarity

- conc. of dominance

- species richness and evenness

what is pielou J

- more focused on evenness than Simpson 1-D

- evenness

- relative abundance

- proportional distribution

- greater sensitivity to richness

Fresh cheeses

chevre, ricotta, mozzarella, and feta

soft bloom cheeses

brie, camembert

semi-firm/firm cheeses

cheddar, manchego, gouda

blue cheeses

fourme d'ambert, gorgonzola dolce

washed rind

taleggio, raclette

bacteria involved in cheese

- lactobacillus, leuconostoc or lactococcus

- sometimes followed by rennet (enzyme cuts proteins in milk, making better curd)

- drain the whey, keep the curd

- lactose (milk sugar) into lactic acid

info on fresh cheeses

- the "first cheeses"

- young, wet, more milk and grass flavors

- no hard rings, added rind components

- lactose into lactic acid -> coagulation