Fermentation: Microorganisms and Processes

• Fermentation is a process where microorganisms cause chemical changes in food or drink.
• This results in the production of substances like gas (CO2), alcohol (EtOH), and other organic compounds (lactic acid, acetic acid).
• These products alter the food's flavor, texture, and stability.

Microorganisms in Fermentation

• Microorganisms generate desired flavors by breaking down macromolecules into smaller molecules.
• They create desired textures through CO2 production.
• They extend shelf life by producing acid, which reduces pH.
• Microorganisms use nutrients for energy, homeostasis, growth, and replication.

Key Microorganisms in Fermented Foods

• Fermentative yeasts, lactic acid bacteria (LAB), and acetic acid bacteria (AAB) are key microorganisms.
• Enzyme-producing molds and bacteria hydrolyze macromolecules.
• Probiotic bacteria offer health benefits by competing with GI microbes and producing antimicrobials.

Catabolism and Anabolism

• Catabolism breaks down complex molecules into simpler ones, generating energy.
• Anabolism synthesizes complex molecules from simpler ones.

Cellular Respiration

• Cellular respiration is the metabolic breakdown of nutrients to create energy.
• It can be aerobic or anaerobic, consisting of glycolysis, the citric acid cycle, and oxidative phosphorylation.

Glycolysis

• Glycolysis is an anaerobic, multi-step reaction that converts glucose to pyruvate and NADH.

Citric Acid Cycle

• The citric acid cycle doesn't use oxygen directly, but it requires oxygen to continue.
• It processes pyruvate into NADH.

Oxidative Phosphorylation

• Oxidative phosphorylation is aerobic.
• NADH donates electrons, which pass through protein molecules.
• Oxygen is the final electron acceptor.
• Protons are sent across a membrane, driving ATP synthesis.

Alternative Fate of Pyruvate - Ethanol Fermentation

• Yeast fermentation yields ethanol and CO2.
• Glycolysis produces pyruvate, which is then converted into ethanol and CO2.
• Pyruvate decarboxylase reduces pyruvate to acetaldehyde and CO2.
• Alcohol dehydrogenase reduces acetaldehyde to EtOH.

Alternative Fate of Pyruvate - LAB Fermentation

• Lactate dehydrogenase converts pyruvate to lactate, regenerating NAD+ for continued glycolysis.
• NAD+ and NADH are cycled between glycolysis and fermentation in the homofermentative pathway.

Homofermentative

• In homofermentative lactic acid bacteria, glycolysis converts glucose to pyruvate.
• In the absence of oxygen, pyruvate is converted to lactic acid by lactate dehydrogenase.

Heterofermentative

• In heterofermentative lactic acid bacteria, the phosphoketolase pathway starts with glucose to yield CO2 and xyulose-5-phosphate.
• This is converted to produce lactic acid along with acetic acid/EtOH.

Alternative Fate of Pyruvate - LAB production of Acetate

• Pyruvate dehydrogenase activity yields acetate with CO2 liberation.
• Pyruvate-formate lysate activity yields acetate and EtOH.
• Pyruvate oxidase also produces acetate.

LAB Metabolic Activity

• LAB synthesize EPS, that contributes to yogurt's properties
• They metabolize casein, contributing to cheese flavor.
• They ferment citrate to produce diacetyl.
• They process propionate to propionate, acetate, and CO2, contributing to cheese flavor and texture.

LAB and Sugar Transport

• PTS, symport, and ABC systems help LAB import sugars.
• PTS transfers a phosphate group from PEP to sugar and cleaves disaccharides.
• Symport uses an ion gradient to transport sugar into the cell, coupled with a permease enzyme.
• ABC systems use proteins to capture and translocate sugar solutes across the cell membrane.

Acetic Acid Bacteria (AAB) Production of Acetate

• AAB converts EtOH to acetaldehyde using alcohol dehydrogenase.
• Acetaldehyde is then converted to acetate.

Vinegar Production

• Yeast converts sugar to EtOH and CO2.
• AAB converts EtOH to acetic acid.

Indigenous Cultures

• These use microorganisms naturally present on the raw food.

Backslopping

• This involves adding a portion of a successful batch to a new batch.

Starter Cultures

• Specific microorganisms in pure cultures are added to raw food to dominate the natural microbiota, enabling controlled fermentation.
• Starter cultures allow for consistent fermentation, predictable timing, product quality, and safety.

Properties of Starter Cultures

• Starter cultures consistently produce desired flavor and texture.
• They have controlled and predictable fermentation rates.
• They tolerate food properties, fermentation processes, and handling stresses.
• They are phage-resistant and tolerant of preservation methods.
• They are compatible with co-cultures.
• GRAS (Generally Recognized as Safe) status requires identification as a specific organism.

Bulk Cultures

• These are smaller-scale inoculations to increase starter culture populations before larger production.

Direct-to-Vat

• This culture must be concentrated and stable for preservation.
• More convenient than bulk cultures but can be costlier.

Starter Culture Vulnerability

• Bacteriophages are viruses that infect bacteria.
• They have lytic (kills bacteria) and lysogenic (incorporates genetic material) life cycles.

Bacteriophage Control

• Sanitation chemicals inactivate microorganisms.
• Exclusion keeps starter cultures isolated from the rest of the food processing.
• Phage-resistant cultures block stages of the bacteriophage lytic cycle.

Bacteriocins

• Antimicrobial peptides are produced by some bacteria that are antagonistic to other bacteria.
• These can be released by live bacteria or added directly to food.

Key Microorganisms in Fermented Foods

• Yeast: Utilizes food sugars to produce CO2 gas and EtOH.
• Saccharomyces cerevisiae is most important for many food fermentations.

Lactic Acid Bacteria (LAB)

• These bacteria do not form spores, can grow with or without oxygen, produce lactic acid, and have various tolerances for acid, salt, and temperature.
• Lactobacillus, Lactococcus, Leuconostoc, and others are included.

Acetic Acid Bacteria (AAB)

• These bacteria do not form spores, are obligate aerobes, and produce acetic acid from EtOH.
• Acetobacter and Gluconobacter are included.

Gram-Positive Bacteria

• Other Gram-positive bacteria produce lactic acid but aren't included in LAB grouping.
• Bacillus sp., Listeria sp., and Bifidobacteria sp. are examples.

Lactobacillus

• This is a large and diverse genus.
• They are mesophilic, aerotolerant, and acid-tolerant (diverse).
• Found ubiquitously except in extreme environments.
• They are indigenous to the starting product or used as starter cultures.
• Examples in dairy, sausage, sourdough, pickles, and sauerkraut.

Lactococcus

• Found naturally in raw milk.
• Starter culture for many hard cheeses and cultured dairy products.
• Homofermentative.
• lactis subsp. is most important for food fermentation.

Streptococcus

• Diverse genus (pathogens, commensals of intestinal tract, fermentation species).
• Used primarily for yogurt and cheese production.
• Homofermentative.
• More heat tolerant than Lactococcus but its nutrient requirements more demanding.
• Growth better with free amino acids since only weakly proteolytic.

Leuconostoc

• Associated with plants and dairy products, dependent on sugars metabolized.
• Mesophilic temperature range growth.
• Heterofermentative.
• Important for sauerkraut for initial drop in pH and other LAB growth.
• Important for dairy fermentations for its production of diacetyl.

Pediococcus

• Tolerant of high acid and high salt.
• Homofermentative.
• Naturally present on raw vegetables.
• May be added to meat to produce fermented sausages.

Oenococcus

• Important species for wine.
• Heterofermentative.
• Most ethanol-tolerant of LAB.
• Tolerant of low pH.
• Can de-acidify wine for smoother taste.
• Uses malolactic fermentation to decarboxylate malic acid to lactic acid.

Tetragenococcus

• Homofermentative.
• Mesophilic temp, neutrophilic pH, halophilic salt conc.
• Tolerance of salt not necessarily mirror tolerance to high sugar concentration.
• Important for soy sauce and salty fish sauces.

Acetobacter and Gluconobacter

• Make acetic acid via oxidation of ethanol.
• Important for vinegar.
• Can be a spoilage agent for wine, beer, cider.

Bacillus

• Can be spoilage agents, pathogenic varieties as well.
• Used for natto.

Kocuria, Micrococcus, Staphylococcus

• Provide aroma compounds by hydrolysis of lipids and proteins.
• Used for manufacture of dry fermented sausages.

Bifodobacterium

• Produce lactic acid and acetic acid and are added to fermented dairy products for probiotic benefit.

Brevibacterium linens

• Cheese ripening for production of yellow-orange-red pigment of cheese.
• Contributes to cheese flavor.

Propionibacterium

• Dairy habitat.
• Produce propionic acid, acetic acid, CO2.
• Used to create 'eye' (holes) of Swiss-type cheese.

Acids and Carbon Dioxide

• Lactic acid: tartness and texture to fermented dairy products
• Acetic acid: vinegar flavor
• Diacetyl: buttery flavor of fermented dairy products
• Carbon dioxide: textural properties to food

AAB Oxidative Reactions

• Conversion of ethanol to acetaldehyde by alcohol dehydrogenase activity.
• Conversion of acetaldehyde to acetate by aldehyde dehydrogenase activity.

AAB Multi-Step Process

• Starts with a food source of nutrients.
• If it is simple sugar, it is ready to use.
• If starch is present, it is saccharified.
• Yeast converts sugar to ethanol and CO2.
• Acetic acid bacteria convert ethanol to acetic acid.

Casein Proteins

• Casein proteins constitute approximately 80% of total protein in milk.
• They are precipitated from acidified milk to form curd.

Whey Proteins

• Whey proteins make about 20% of total protein in milk.
• Liquid portion that separates from curd.
• Also contains lactose, water-soluble vitamins, and some minerals.

Yogurt Processing

• Fermentative starter cultures produce lactic acid, which reduces pH and coagulates milk proteins.

Yogurt Labeling

• Yogurt is labeled as 'Live and Active Cultures' if containing 10^8 CFU/g bacteria.

Cheese Manufacture Steps

• Milk, curd formation, cut + cook, salt, and aging

Curd Formation

• Acid production by fermentative bacteria
• Addition of chymosin enzyme (rennet) to alter casein protein surface for casein protien aggregation

Aging (Ripening)

• Ripening develops flavor by enzymatic breakdown of lipids and proteins.
• Longer aging periods result in more complex flavors and firmer texture.

Cheese Curd Chemistry

• Curd is clumping of casein proteins.
• Bacteria or enzymes change the protein micelle surface to encourage clustering.

Chymosin

• Enzyme added to milk to make cheese.
• Originally sourced from stomachs of calves, now produced by genetically engineered MOs.
• Culture may be added first to allow slight pH drop which favors enzymatic activity of chymosin.
• Mesophilic and thermophilic cheese starter cultures exist.

Cheese Aging and NSLAB

• Non-Starter Lactic Acid Bacteria
• Reinstate bacteria associated with indigenous flora inactivated by pasteurization.
• Out-compete indigenous bacteria associated with development of off-flavors.

Cheese Aging

• Compounds are released from breakdown of macromolecules (protein, carbs, lipid).
• Flavors are formed during bacterial metabolism by free enzymes.
• Microbial populations fluctuate

Whey Usage

• Some cheese made from whey (ricotta).
• Can be concentrated and used as ingredient to improve texture of other foods.
• Can be a replacement for carb gums.
• Promotes water retention in some meat products.
• Acts as a fat substitute.

Fermented Meat Processing Steps

• Grind to desired coarseness
• Mix in ingredients
• Stuff in casing
• Ferment
• Dry to final moisture of 35% to 50%

Ingredients in Fermented Meats

• Sugar: a nutrient source for fermentative microbes
• Salt: flavor, solubilize muscle proteins, microbial inhibition
• Culture: Pediococcus spp., Lactobacilli spp.
• Curing agent: nitrite or nitrate for pathogen control
• Ascorbate: antioxidant

Nitrite and Nitrate Salts

• Nitrite works directly as curing agent.
• Nitrate must be converted to nitrite by microorganisms.
• Celery juice/powder naturally contains nitrate and may be used with nitrate-reducing MOs to avoid nitrite label.

Functions of Nitrate

• Maintains meat color (interacts with myoglobin protein)
• Antimicrobial agent (protects against outgrowth)
• Aantioxidant (ptotects against rancidity

Ripening Adjuncts (Meat)

• Fungi growth provides flavor through production and secretion of proteases and lipases to meat that impart flavor.
• Similar concept to cheeses.

Defects of Meats

• Overgrowth of spoilage MOs (Pseudomonas sp.).
• LAB may produce exopolysaccharides resulting in slimy appearance, hydrogen peroxide that can lead to rancid flavors and pale coloration of meat.

Fermented Vegetables

• Usually no specified starter cultures.
• Indigenous microorgansims used.
• Conditions are established that favor growth of fermentative MOs and suppress pathogens through salt application.

Sauerkraut Processing Steps

• Remove outer layer
• Shred cabbage mechanically
• add salt
• cover and compress cabbage to create anaerobic conditions and prevent contamination
• Ferment
• Package for final storage

Functions of Salt Addition to Sauerkraut

• Inhibits spoilage MOs.
• Helps water and water-soluble nutrients defuse from plant to create brine.
• Inhibits enzymatic activity.
• Provides flavor.

Sauerkraut Fermentation

• Stage One is heterofermentative and produces gas.
• Salt favors Leuconostoc mesenteroides in anaerobic conditions.
• Leuconostoc mesenteroides produces lactose, and acetic acid.

Sauerkraut Fermentation - Stage Two

• The L. mesenteroides population declines due to increased acidity.
• The acidic conditions favor Lactobacilli spp., and Pediococcus spp.
• The production results in a final acidity of 1.7% and pH 3.5.

Bread Fermentation

• Differs from other fermented foods by being a more perishable end product.
• Converts stable raw product (wheat grain) to more perishable finished product (bread).
• Fermentation byproducts mostly not retained as yeast inactivated and EtOH is cooked off.

Cereal Grains

• Grains are derived from edible seed portion of grasses grown as agricultural crops.
• Can be milled into flours for use in a baked goods, cereals or snacks.

Wheat Grain Makeup

• Bran, endosperm, and germ

Bran Makeup

• Fibrous and very high in cellulose.

Endosperm Makeup

• Large Inner Portion of Cereal Grains
• Composed primarily of starch and some protein.

Germ Makeup

• Embryo: fat, small amount of protein, B vitamins - esp thiamine and riboflavin

Wheat Flour Protein

• 8-14% of wheat flour (~85% gluten)

Gluten

• Gliaden and glutenin complex that develops in wheat flour.
• Water creates elastic protein structure via entrapment of gas

Gliaden

• Protein fraction of wheat gluten that is compact, elliptical, sticky and fluid.

Glutenin

• Protein fraction of wheat gluten that is fibrous, elongated, and elastic.

Wheat Flour Carbohydrates

• 75% of wheat flour, primarily starch.

Starch

• Complex carb comprised of two fractions, amylose and amylopectin - both chains of glucose with different linkage configurations
• Broken into glucose and maltose to provide source of sugar for yeast

Amylose

• Straight chains of glucose
• 20-25% of starch