FST 104 Final study

What is the common end product made by homo and heater fermenters

LACTIC ACID homo end product: lactic acid hetero end product: make other end products like acetyl aldehyde and ethanol

Stomach

Acidic, supposed to kill everything and prevent us from getting sick but the stomach has 10^2 microbes in every ml of fluid When we swallow we tend to swallow air (in the stomach there's a whole bunch of air) Good for aerobic environments

1. 6 fundamental food characteristics

Acidity Water activity Redox potential Temperature Chemical constituents Other

Each of these regions has different bacteria that are present in it Each of these environments require different adaptations for bacterial survival

As you move down lactobacilli and streptococci deplete the oxygen

Guidelines for fermentations

LAB fermentations occur in the presence of high soluble sugars and low oxygen, results in high Other scenarios: (aerobic conditions) with low or moderate amounts of sugar and mainly high MW compounds

Safety Concerns

Most food pathogens belong to Enteric and Endospore bacterial groups Spoilage conditions which encourage either of these groups might also be a safety concern

Good manufacturing practice

Specific for the food Set definitions on food manufacturing procedures

simple type of complex fermentations

Tempeh: soybeans fermented by rhizopus Inoculated with mold on banana leaves Benefits Soy protein becomes more digestible Increased amount of vitamin b12 (from bacteria) Nutty taste and ammonia

Progression of sauerkraut graph

x axis is time y on left is log bacterial count y right is percent of lactic acid (expect it to go up and pH to go down)

Vanilla

Seed pods of an orchid plant "cured" for several weeks: LAB hydrolyze glucoside, glucovanillin, to form vanilla

Variables that influence LAB metabolism end products

1. Carbon source (sugars) available 2. Presence or absence of oxygen 3. Presence of citrate (pyruvate) 4. Presence of other organic compounds 5. pH and temperature

5 strategies for predicting spoilage

1. Identifying food characteristics 2. Microbes likely to be present 3. Microbes capable of growing 4. Fastes growing group (FGG) 5. Effects caused by the FGG

1 step fermentations

1. Starting material is soaked and heated - soften the bean and increase aw and liberate some sugars and nutrients out of the bean (make it more accessible to microbes to grow) 2. Inoculum (starter culture) is added - molds and bacillus digest proteins and starch in foods 3. Incubate for 1-2 days Advantage: increased digestibility in the human gut BUT pH is not lowered and product is susceptible to spoilage

Characteristics of probiotic strains (4)

1. isolated from the human gut (does not always happen) 2. survive in food products and in the stomach and small intestine 3. not resistant to antibiotics and are otherwise safe (GRAS - generally regarded as safe) 4. provide demonstrable and specific health benefits

Characteristics of prebiotics

1. remain intact until reaching the large intestine -resistant to gastric acid -undigested by mammalian enzymes -not absorbed 2. digested/fermented by selected groups of bacteria in the large intestine -stimulates growth or activity of those selected bacteria and NOT OTHERS! 3. confer benefits to human health

Yogurt

40 degrees celcius (above body temperature) Two species we need to have in order to make yogurt *Streptococcus thermophilus - fast growing, produces lactic acid, acetic acid, and acetaldehyde **Lactobacillus delbrueckii ssp bulgaricus -proteolysis 6 hours high temp and final result: 2% lactic acid, pH 4.6 streptococcus grows quickly and then stalls out because its runs out of somethings while the lactobacillus chugs along quickly -streptococcus runs out of amino acids which it needs for growth and the lactobacillus releases aa so the streptococcus can grow again

Radiation doses for lethal terminology for bacteria uses "D-values" Example: D=6 kGy

6 kGy will achieve a 10-fold (1 log) reduction in the number of living bacteria instead of time it's in kGy (amount of energy exposed)

Gut microbes and disease

Alterations in the intestinal microbiota are correlated with disease

Human body is colonized by 10^13-10^14 bacteria cells

But there are only about 10^13 human cells There is 100 times as much microbial genetic information Heavy area of microbe is our digestive tract

Meat and vegetable fermentations

Chop - release nutrients for the microbes to grow (glucose) Salt - leaching of sugars; protection from spoilage aw goes down prevents some microbes from growing Spice - microbial growth promoters (Mn2+) or inhibitors Pack - microaerophilic/anaerobic environment, reduce the amount of oxygen available

Coffee

Coffee made from the beans (cotyledons) which are surrounded by pectin Pectinolytic bacteria degrade the pectin (erwinia) and lactic acid bacteria grow

X-rays

Created through electrons passing through metal Very short wavelengths that damage the cells 10^-12-10^-10

Radiation

Creates free radicals inside cells Hydroxyl ions Damages DNA - double stranded breaks Cells cannot replicate and die

How can we influence the microbiota in us

Diet Age Environment Antibiotics

Major genera of LAB: phylogeny

Division: Firmicutes Class: Bacilli Order: Lactobacillales Lactobacillus Streptococcus Lactococcus - hard cheeses Leuconostoc - saurekraut Pediococcus Oenococcus

2. The presence of air (oxygen)

Does not produce energy but rather restores NAD to the cell Less lactic acid is made RESULT: higher pH and more pyruvate (because you don't need to convert it to lactic acid) Oxygen used directly to reoxidize NADH -cells also have to have ways to deal with excess hydrogen peroxide Both enzymatic or not (auto oxidation)

Reduce pyruvate to lactate and doing so they regenerate oxidized NAD for glycolysis

If the cell has other ways to regenerate NAD they will do it

What is fermentation

Energy metabolism without an electron transport chain - relies on substrate level phosphorylation Large scale growth of microbes for specific products (whether oxygen is present or not) *Process of making a food in which the characteristic properties of the food are the result of extensive microbial growth

Rennin (chymosin)

Enzyme found in a calf's stomach Rennin is now commonly produced for cheese using recombinant E. coli, molds or yeast Cleaves between phenylalanine and methionine bonds in k-casein Optimum pH is 5.5 Result: precipitation of casein

Other LAB end products

Ethanol CO2 Acetic acid vinegar flavor Acetaldehyde yogurt flavor Diacetyl butter flavor Formic Acid fruit 2,3-Butanediol sweet

Food safety modernization act (2011)

Finally solid rules Company is responsible Prevention not just correction

Cheese

First cheese: 6000 BC Over 2000 types of cheese in the world In US we only make Cheddar and Mozzarella California is number 1 when it comes to milk production 2nd largest cheese producer Milk --> inoculate starter cultures and add rennin (aka chymosin-degrades casein) --> casein (precipitates at pH 4.5 (without chymosin) and/or by rennin)

Bread fermentations

Flour water and starter culture Standard bread: Yeast (Saccharomyces cerevisiae) makes CO2 and causes the dough to rise and gives bread the flavor Sourdough: Distinctively sour taste Acetate 10 X higher concentrations that in regular bread No added sugar and longer cooler rising cycle

3. Determine which microbes can grow

Food characteristics + Growth requirements Microbial growth

Health claims

Foods and dietary supplements: PERMISSIBLE: statements relating the food or supplement to the normal functioning of the human body (structure/function claim) NOT PERMISSIBLE: role of product in prevention, treatment, cure, diagnosis, or mitigation of disease (health claim)

LAB

Grow throughout foods (many are oxygen tolerant) Saccharolytic fermentative metabolism: typically consume mono- and di- saccharides (glucose, lactose, sucrose) *not going for the proteins and fats Types of energy metabolism 1. homofermentation 2. heterofermentation 3. respiration

Hazard Analysis Critical Control points COMPANIES HAVE TO HAVE THESE PLANS

HACCP Most real - how we can control microbes in food where are the problem points - where can microbes get in and how can we control them best 6 principles: DON'T MEMORIZE THEM

Enterics ecoli salmonella shigella

Habitat: Human/animal intestine -erwinia is an exception (plants) some erwinia can degrade pectin and produce something that looks like a mold but it's actually a bacteria problem Oxygen requirement: Facultative anaerobes Tolerance: pH 4.5 aw 0.94 -some psychrotrophs (enterobacter, proteus, erwinia) -NO thermophiles Growth rate: moderate to high growth Unique: prefers sugars, but can consume proteins protein degredation: "rotten smell" sugar fermentation: organic acid -erwinia produces pectinase (soft rot) Typical foods: all foods handled without good sanitation erwinia on vegetables

Staphylococcus Aureus makes heat stable toxin

Habitat: human/animal skin Oxygen requirements: facultative anaerobe Tolerance: pH 4.5 low aw 0.86 -7 to 48 degrees celcius for growth (enterotoxin: 10 to 46 degrees celsius) Most similar to the "endospore forming" group Produces lipases and proteases Similar growth rate But does not form endospores

Endospore forming bacteria

Habitat: mainly from soil Oxygen requirements: facultative and strict anaerobes Tolerance: pH 4.5 (or 3.7) aw -.94 some thermophiles some psychrotrophs Growth rate: grow slower than some other food associated bacteria Unique: form endospores that can germinate (after heating the foods) extracellular hydrolases (proteases/amylases) Typical foods: cooked and canned foods, herbs and spices moist baked foods foods high in protein and carbohydrates

Yeasts

Habitat: plant microbiota Oxygen requirements: aerobes and facultative anaerobes Tolerance: low pH 2.0 low aw 0.8 some are psychrotrophs (NO thermophiles) Growth rate: slow, but faster than molds Unique: most require> 1% sugar concentrations facultative anaerobes grow throughout the food Typical foods: fruit juices/jams fermented foods

Lactic acid bacteria

Habitat: plant/animal microbiota Oxygen requirements: facultative anaerobes aerotolerant anaerobes Tolerance: low pH 3.5 moderate aw 0.9 psychrotrophs and thermophiles Growth rate: grow slower than some other food associated bacteria Unique: fermentative, important in food fermentation mainly uses soluble sugars; produces lactic acid Typical foods: milk, meat, juices, cut fruit and veggies

Pseudomonas

Habitat: soil (runoff), water, plants Oxygen requirements: facultative anaerobes Tolerance: pH 5.5 aw 0.97 fast growing psychrotrophs Growth rate: grow very fast under aerobic conditions Unique: grows well on sugars, proteins, and fats -extracellular proteases and lipase -bad odors from protein/fat breakdown Typical foods: low sugar, non acidic (pH 5.5+) cold foods refrigerated meat, non fermented dairy or vegetables

Molds

Habitat: soil, plants, spores in air Oxygen requirements: strict anaerobes, grow on SURFACES of food Tolerance: low pH 1.5 low aw 0.6 some are psychrotrophs (NO thermophiles) Growth rate: slow growth compared with bacteria Unique: extracellular hydrolases -metabolize organic acids (raise pH) -decompose macromolecules (proteins) to subunits Typical foods: fruits, fermented food (dairy/vegetables), breads, preserves

Clostridium difficile infections

Healthy colon- antibiotics are good, they kill the bacteria we don't want there But they can't exclusively target it to the one you want to kill, so you might kill the good as well as the bad bacteria Clostridium is strictly anaerobic but they can make spores which can survive in the air If you consume those spores and they reach your small intestine they can move in and cause all sorts of issues Now you have the wrong set of organisms living in your gut But the spores aren't killed by the antibiotics - vicious cycle

LAB continued Tolerance

Hugely genetically diverse Stress tolerant salt:>5% NaCl (aw 0.9) acid: pH 3.5 temperature: psychrotrophs (some can grow in the refrigerator) and thermophiles alcohol: tolerate 12-13% ethanol oxygen: facultative anaerobes aerotolerant anaerobes

Environmental limits of food pathogens

In general, pathogens do NOT grow: below aw of 0.85 (S. aureus) below pH of 4.5 (pathogens will not survive a low pH when they are growing - they can't keep their internal pH at neutral) Above a temperature of 60 degrees celsius (C. perfringens) Pathogens are either facultative anaerobes or obligate anaerobes Therefore, no redox potentials are safe from pathogens except extremely high (ozone) or low redox

4. Citrate

In milk, metabolized through enzymatic reactions --> pyruvate and makes other products If citrate is available there would be another way to regenerate NAD through reduction of other compounds One of the end products is diacetyl If citrate is available you get higher dactyl (butter flavor) The quantity of the butter flavor depends on the pH of the milk Permease transporter is pH sensitive optimum pH is 5-5.5 When pH <5 less citrate is taken up and less diacetyl is produced

Gamma rays

In the wavelength of 10^-14-10^-12 nm Spectrum of light ultraviolet radiation Gamma rays are the smallest

What are the changes that happen during fermentation

Increased vitamin production Fermentation detoxifies the lactose in milk - so people who are lactose intolerant can eat it (in general) Foods rich in proteins fats and starches typically fall into complex fermentations -often involved in succession in microbes -outcome is changes in the properties of the food and flavors of the food

USDA agencies

Inspect the raw materials -plants and animals free from pathogens and pests

Ways cells regenerate NAD+

Lactate synthesis Respiration Oxygen metabolism Reduction of other cell compounds (pyruvate to 2,3 butanediol)

Lactic acid

Lactic acid is the major end product of LAB metabolism -made from pyruvate as a way for the cell to keep it's redox balance -for us lactic acid lowers the pH of the environment of that organism -it's not so temperature sensitive, active over a broader range of temperatures and this might get more or less LA made depending on the temperature which has a mild sour taste)

Camembert and Roquefort cheese (MESOPHILIC)

Lactococcus lactis Leuconostoc cremoris Camembert: penicillium camemberti (white cheese mold spread on the surface) Roquefort: penicillium roqueforti (a mold found in soil of local caves in roquefort-usr-soulzon) Traditionally - placed cheese in rooms loaded with mold spores, the mold is directly inoculate in pins Contributions of mold: peptides, ammonia, fatty acids = flavor Degrade organic acids (lactic acid) Increase pH from 4.8 to 7 (susceptible to spoilage and pathogens - listeria)

Organisms names and associations to know Lecture 24 Find the foods these organisms are in

Lactococcus lactis and Leuconostoc mesenteroides Streptococcusthermophilus and Lactobacillus delbruckeii spp bulgaricus Propionibacterium PenicilliumcamembertiandPenicilliumroqueforti SaccharomyceskefirandLactobacilluskefir

Fermented fruits and vegetables

Many fruits and vegetables can be fermented for food: Cabbage Cucumbers Olives Cocoa Coffee Tea Vanilla Artichokes Carrots Celery Tomatoes Peppers Beets Onions Acetic acid (vinegar) and lactic acid are sometimes added as a substitute for the growth of LAB, this is true for most commercial pickles sold today

Fermented meats: salami and pepperoni

Meat (pork or beef) Grind Mix (with salt, glucose nitrate ascorbate spices starter culture) Stuff in casing (reducing exposure to air) Ferment 2-4 days Dry/smoke/cook moist/semidry/dry

Categories of LAB in cheese

Mesophilic: Topt= 20-35 degrees celcius [example: cheddar and brie colby gouda] room temp Thermophilic: Topt= 37-45 degrees celcius [example: swiss provolone mozzarella] above room temp BOTH MAINLY HOMOFERMENTATIVE

5. Influence on other microbes

Microbes can make the environment more suitable to other microbes through: Increasing pH (metabolizing acids) Digestion of high molecular weight barriers (proteins) Mold growth -->(metabolize acids/break down high MW barriers) Bacterial growth

4. Identify which microbes grow fastest

Microbes that grow fastest usually dominate Microorganisms compete for nutrients in food If two microbes grow at the same rate, the one with the highest initial amounts will dominate If other microbes are present others will usually come... succession

Large intestine

Microbial hot house, as many as 10^12 bacterial cells per ml

3 causes of food spoilage

Microbial spoilage Autolysis: enzymes can cause undesirable changes in color, texture and flavor of uncooked foods Other factors: low temperature injury (refrigerated cucumbers)

Dairy fermentations Basic ingredients

Milk + LAB 1. Artisanal (spontaneous) fermentations -do not add any bacteria (use those already in milk) -10^3-10^5 cells per milk, not sterile -leaving a lot up to chance, must have enough LAB in milk for it to ferment properly 2. Industrial fermentations: starter cultures -instustrial LAB cultures are added to (pasteurized) milk to start the fermentation -rapidly acidify the milk to prevent growth of unwanted things 3. Industrial fermentation: adjunct cultures -bacteria that grow after the starter bacteria (either inoculated or naturally present after pasteurization)

Cheese (continued)

Milk --> inoculate starter cultures and add rennin (chymosin) --> casein (precipitates at pH 4.5 and or by rennin) Next is either whey (liquid) which is incubated to pH 4 and pressed to ricotta cheese Or casein (curds) are pressed to make soft cheese and then aged to make hard cheese

What are the outcomes of food fermentations

Milk makes cheese kefir and yogurt What has changed from the milk to make these other products? -sugar amounts -change in nutrient content -lower redox -increased microbe number A lot of different products

Dairy Fermentation

Milk pH 6.8 LAB grow and are reducing the pH through production of lactic acid pH 5.3 for cheese pH 4.6 for yogurt Dairy fermentations is mainly homofermentative LAB

simple type of complex fermentations II

Natto: soybeans fermented by B. subtilis Soybeans soaked and heated, inoculated, wrapped, and incubated Traditionally made on rice straw Benefits - soy protein becomes more digestible Unique flavor and texture (b. subtitles exopolysaccharide)

2. Determine which microbes are likely to be present

Normal microbiota of plants or animals Natural contaminants -soil -dust -water Handling contamination Human contamination (skin, fecal-oral)

5. Other carbon-metabolism pathways

Not used to regen NAD Other compounds present in milk can result in end products that can benefit us or that we don't want *amino acids just about changing the flavor of the food Methanethiol flavor = garlicy desirable in cheddar

Government regulation of food

Objectives -inform consumers about food nutritional content - what is a healthy diet and what food should we be eating more of, what's on the label -ensure safety of food -prevent fraud Oversight and regulation -depends on where the food is made -what type of food [for who is regulating it]

Kefir

Origin: balkan mountains Beads contain: LAB that ferment lactose to lactic acid Yeasts that ferment lactose to ethanol (2%) and CO2 Lactobacillus acidophilus Lactobacillus plantarum Lactobacillus casei Lactobacillus kefir Saccharomyces kefir

Gut microbiota: common functions

Perform important tasks: Aid in food digestion Produce vitamins and AA Stimulate immune system function Stimulate gut development Protect against pathogens Influence neural development and activity

Sauerkraut fermentation cabbage is chopped, salted and packed

Phase 1. Heterofermentation Leuconostoc mesenteroides - should see gas?? why bc hetero?? Grows at cool temperatures (sauerkraut = 18 celsius) tolerant to O2 and not highly acid tolerant Phase 2. Homofermentation Lactobacillus plantarum Less tolerant to O2, acid tolerant

LAB heterofermentation some only do hetero some only do homo some do both called heterofermentation because we end up with multiple end products

Phosphoketolase pathway Only heterofermenters have phosphoketolase Both phosphorylate glucose and then the pathways diverge Reduce NAD twice to NADH Lactic acid is an end product but they have two of those NADH they have to regenerate back to NAD Payoff - 2 ATP produced net gain of 1 ATP

Food based approaches to deliberately change our microbiota

Probiotics must be living and must confer a health benefit (mostly strains of lactobacillus and bifidobacterium) Prebiotics (selectively fermented) food you consume to improve the activity of beneficial bacteria (native beneficial bacteria) Synbiotics combination of pro and probiotics

Lactic acid bacteria (LAB) So important for most fermentations

Produce (lactic) acid as end-product of metabolism, OF THEIR GROWTH Found on plants, digestive tracts (animals and insects), milk (found in the raw materials themselves) Gram+ Firmicutes - relatives of listeria staphylococcus

How do we study the microbiota

Proper sampling Who is actually there (every cellular organisms require ribosomes universal marker, used to identify organisms --> like a fingerprint) What are they doing - different under different circumstances, first find out what they could be doing [with no arms you cannot juggle] 16 srRNA - what is the expressed RNA in an environment called metatranscriptomics Translation is how you get from RNA to proteins - therefore, you could look at proteins called metaproteomics But to know exactly what happens you must look at the metabolites - is a certain molecule being made or not [metabonomics] Methods using PCR using mass spec we can find out who is there and what they are doing

LAB homofermentation some only do homo some only do hetero and some do both Called homofermentation because we get lactic acid

Really just glycolysis aka Embden-meyerhof-parnus pathway Can consume glucose galactose and a variety of other sugars Payoff phase - 4 ATP produced (net gain of 2) 2 NADH produced NAD+ is regenerated Glycolysis can continue -if the cell just stopped at pyruvate it wouldn't be around, it would be loaded with reduced NAD+ -so they reduce pyruvate to lactic acid which allows the cell to reform the NAD back into the reaction from glycolysis

Short chain fatty acids (SCFA)

Reduce pH of the lumen = make the lumen less hospitable to pathogens Can make us feel full

Cocoa: complex fermentations

Remove the beans and pulp from the pods and dry in heaps on banana leaves Fermentation takes 1 week Variety of organisms involved LAB and yeast growing make LA ethanol and co2 That ethanol is converted to acetic acid by acetic acid bacteria (requires o2) The endospore forming bacteria and molds can then form from the acetic acid

Sourdough

Requires a warmer environment to grow well but not in sourdough so expect diff types of yeast Candida milleri: only consumes glucose does not consume maltose (disaccharide of two glucose molecules) grows at 30 and not 37 degrees celsius Lactobacillus sanfranciscensis is the LAB that is making all the acetic acid It just consumes maltose Strict heterofermenter Requires a special peptide made by candida Strict AA requirements Complementary symbiosis between these two organisms in the bread

Case study on mouse

Set of mice on low fat diet and western diet (high fat, high carb) The microbes on the low fat diet stayed consistent

Tea

Some fermentations occurs during the drying of harvested leaves which contributes to tea flavors -initially still moist where the microbes can grow on the leaves and metabolize the sugars Kombucha tea Fermentation of sweetened tea by yeast LAB and acetic acid bacteria (make vinegar out of ethanol)

Respiration in LAB

Some of them can respire -don't contain a complete TCA cycle or complete ETC -need a little help to perform respiration -Must be given heme or vit k to support the ETC -can use o2 as electron acceptor -or can do anaerobic and use nitrate (pH doesn't go down as quickly) End product will still be lactic acid but the cells can be oxidized through the ETC instead of always reducing it to lactic acid

Food systems

Source Transportation Retail service Home

Swiss cheese example

Starter culture in milk Add rennin (chymosin) Forms curds in 30 min Cut (to introduce oxygen) Scald (50-55 celcius) for 50 min this drys curds Whey removed Pressed Ripened for weeks 10-18 celcius

Soy sauce: complex fermentation

Step 1 Koji Soybeans soaked, heated and inoculated with Aspergillus oryzae (no mycotoxins) Incubate 3 days Results in increases in free sugars, peptides and amino acids (nutrient base for step 2) Step 2 Moromi Koji mixed with 18% salt Inoculated with Saccharomyces rouxii and L delbrueckii or pediococcus soyae incubation for 3 to 6mo

Swiss cheese microbes

Streptococcus thermopiles and lactobacillus helveticus and propionibacterium species (G+) causes acne Proprionibacterium consumes lactic acid: Lactic acid --> propionic acid and acetic acid + CO2 [HOLES]

1. Carbon source (sugars) available

Sugars enter the pathway (from foods) and can be fermented -outcome: what's upstream won't happen When pentoses are available: heaterofermenters can grow and no CO2 is produced When fructose is available to some heterofermenters: mannitol (regenerate NAD to make more ATP), acetate, lactic acid, CO2 are produced No ethanol but extra ATP

Gut microbiota functions not on the slides

Symbiotic and some cases mutualistic producing amino acids we don't produce ourselves Can soften inflammatory response Leaky gut happens when your epithelial cells are loosely packed and things can get between, bacteria can tighten the junction between cells

FECAL TRANSPLANTS

Take microbial community of a healthy person and present it to someone with an open niche, the healthy microbes you would hope to fill in, they would take over first Tremendously effective treatment

3. Other sources of pyruvate

The more pyruvate available to the cell the better off it is because it can grow more quickly, DNA, proteins etc

LAB energy metabolism

The type of metabolism they have, the kind of environment they're in, the temperature they are exposed to will ultimately tell us in the end will we have a yogurt or a cheese, a yummy saurkraut or a terrible one

Stomach ulcer

Ulcers used to be believed to be caused by stress, spicy food but there is actually an organism associated with ulcers

Federal food, drug and cosmetic act

Used for 100's of years Now revamped Put definitions on what is dirty, what is filth what is unsanitary etc Reportable food registry Must report an EHEC if found

Microbial resistance to radiation:

Viruses>bacterial spores>yeasts and molds>G+>G->Insects>Humans

Grays

a measure of energy transferred with an x ray you would be exposed to .5 mGy to inactivate salmonella in chicken with X-rays it must be exposed to 4.5 kGy (a million times more radiation)

Starter cultures

acidify the meat to prevent the growth of less acid tolerant microbes/pathogens psychrotrophic, acid tolerant, salt tolerant (lactobacillus plantarum, lactobacillus sakei pediococcus pentosaceus) Can be surface inoculated with molds - gives white coating, molds are digesting the fats and proteins that LAB don't, gives flavor, also prevents spoilage molds from growing -by eating the organic acids made by LAB this can increase the pH -usually put in a cool environment (refrigerated)

Cocoa (II)

chocolate flavor is developed in two stages: 1. fermentation - chocolate flavor precursors are formed 2. roasting - these precursors react, forming the chocolate flavor There is NO chocolate flavor in cocoa beans without fermentation

Small intestine

even more bacteria 10^3-10^7 per ml the small intestine is where your body absorbs most of the nutrients