16 .Food fermentation basics and dairy products 1 (wild, backslopping, starter culture., homo, hetero, bulk direct vat, yogurt, buttermilk, sourcream,kefir)

Lactic Acid Bacteria (gram positive, fermentative, non sporeforming, make lactic acid, facultative anaerobes and used in many food fermentations)

Many bacteria make lactic acid but the term "lactic acid bacteria" specifically refers to a group of functionally and genetically related bacteria • Gram positive • Produce lactic acid • Fermentative • Catalase negative • Non-sporeforming • Low % G+C organisms • Non-motile • Acid tolerant • Generally, they lack porphyrins, a cytochrome system and oxidative phosphorylation - No respiration - All energy is from fermentation! • All grow anaerobically; most are NOT sensitive to oxygen and can also grow in the presence of oxygen • Limited biosynthetic abilities - have complex nutritional requirements

syneresis (defintion, 3 steps to prevent syneresis)

• *Definition* - The separation of water from the coagulated milk • Results in a pool of water on top of the yogurt • Considered a major defect since it is undesirable by US consumer • *Yogurt manufacturers use three different steps to enhance the water binding capacity of yogurt* - Increase milk solids - Heat treatment - Inclusion of stabilizers

compare process to make yogurt with greek yogurt, soy yogurt, yogurt drinks

*Greek Yogurt* • Yogurt with water removed - Also called "yogurt cheese" - Makes a thicker, creamier yogurt • Texture similar to sour cream - May result in a yogurt with: • Reduced lactose (removed with the water) • Reduced protein (whey is removed with the water) - Some makers actually put the whey back into the yogurt so that the resulting greek yogurt is higher in protein than traditional yogurt (on a per ounce basis) • Increased concentration of solids (fat, casein, etc.) • How do you remove water from yogurt? - Easiest way is with cheesecloth *Soy Yogurt* • Similar strategy as regular yogurt except soy milk replaces dairy milk • Problem: lactose is the primary sugar in milk whereas the primary sugars in soy milk are raffinose and stachyose - Traditional starter cultures can not ferment raffinose or stachyose - Solutions • add a fermentable sugar - Sucrose is often used • change the starter culture - Select for strains with the desired activity *Yogurt Drinks* • Stirred yogurt with a low viscosity • Yogurt with two main changes: - post-fermentation agitation - No addition of milk solids or stabilizers • Remember that Non-fat yogurt will typically have >12% milk solids • Non-fat yogurt drinks will have ~8.5% milk solids

Detail of the following yogurt fermentation steps - Milk treatment Yogurt mix Heat treatment

*Milk treatment* • Yogurt can be made from skim, reduced-fat or whole milk - Important to use good quality milk that is free of antibiotics and other inhibitory substances • Add nonfat dry milk to increase the total solids to 12% to 15% - Skim milk has ~8.5% solids - Can also increase the % solids by concentrating the milk by evaporation (less common) • Other ingredients including stabilizers can be added - Gums and starches: • Carrageenan, pectin, gelatin *Yogurt mix* • Usually, the yogurt mix is homogenized if it contains fat - Homogenization is unnecessary in a non-fat yogurt - Some specialty yogurts producers make a unhomogenized, cream-onthe-top whole milk yogurt • Homogenization - the process of ensuring a uniform composition and stable structure throughout a product *Heat Treatment* • In US, yogurt is required to be made using pasteurized milk • However, the yogurt mix receives a heat treatment far beyond pasteurization - Pasteurization • 71.7°C for 15 seconds - Typical yogurt treatment • 85°C for 30 minutes Purpose of Heat Treatment • Pasteurizes the yogurt mix - Well beyond typical milk pasteurization • Denatures nearly 100% of major whey proteins - Increases water binding which results in less syneresis

three methods for starting fermentation

*Wild fermentation -* Relies on indigenous microorganisms present in raw material - Also known as "natural fermentation" *Backslopping -* Transfer a portion of a successful fermentation to fresh raw material to initiate a fermentation *Starter culture -* Uses pure cultures of microorganism(s) to initiate fermentation

advantages and disadvantages of each method for starting a fermentation (natural, backslopping and starter cultures)

*Wild fermentation* Advantages - - Natural - No detailed knowledge about the fermentation organism(s) is required - Can be useful for very complex (meaning many different microorganisms) fermentations -Easy -no need to add microbes or fancy equipment -safe product -resistant to phage Disadvantages - To be successful, the "correct" microorganisms need to be present along with proper conditions - Consistency (quality, time, etc.) - Safety -Slight batch to batch variations -complex microbial successions *Backslopping* Advantage - dont need to start from scratch Disadvantage - Successful first fermentation is necessary - contamination over time *Starter Cultures* Advantage - Safe and consistent product - predictable time schedules Disadvantage -Phage contamination -expensive

List four non Lactic Acid Bacteria (LAB) used in food fermentations. List the correct food for each of the non LAB

*• Acetobacter, Gluconobacter and Gluconoacetobacter* >Acetic acid-producing - Used in the manufacture of vinegar - Also a spoilage issue for ethanol containing products (wine, beer, etc.) - Make acetic acid via oxidation of ethanol • Gram - - Only gram negative bacteria used in the manufacture of fermented foods • Obligate aerobes *• Bacillus* Ubiquitous in foods - Benign contaminants - Spoilage agents - Food poisoning • Some strains of Bacillus subtilis are used for the production of natto - Fermented soybean product *• Brevibacterium* Gram +; strict aerobes • Brevibacterium linens - Bacterial, surface-ripened cheeses • Limburger, Muenster - B. linens produces a yellow-orange-red pigment - B. linens is able to hydrolyze proteins and contributes to cheese ripening and flavor development *• Propionibacterium* • Gram + • Anaerobic to aerotolerant • Generally associated with two habitats - Skin - Dairy products • Used in the production of Swiss-type cheeses - Metabolic end-products • Propionic acid • Acetic acid • Carbon dioxide - Also proteolytic and lipolytic

compare and contrast cup style and swiss style yogurt

*• Cup-Style* - Mix is inoculated with culture and is pumped immediately into cups - If the yogurt is to contain fruit, the fruit is added to the bottom of the cup prior to the dispensing of the mix and culture - Fermentation occurs in cups - Consumer must do the stirring and mixing to incorporate the fruit throughout the product *• Swiss-Style* - Fermentation occurs in a vat - After fermentation, the vat is cooled, flavorings added and the yogurt is pumped into containers **post fermentation** • Fermentation is complete when the target acidity is reached • Yogurt is then quickly cooled to below 4°C - Cooling is required to arrest the fermentation - During the cooling period, the pH may continue to drop by an additional 0.2 to 0.3 pH units • Cup-set yogurts are carefully moved to coolers • Swiss style yogurts are cooled in the vat, then mixed with fruit, and then filled into cups

General procedure for making yogurt (steps, organisms, types)

- Yogurt constitutes >50% of the cultured dairy product market in US *Swiss Style* Milk(Standardize-nonfat,lowfat,whole, nonfat milk solids, stabilizers, other ingredients) ---> yogurt mix (homogenize) ---> heat treatment (85 degrees Celsius for 30 minutes) ---> cool (40 to 45 degrees celsius) ---> inoculate (2.0 to 2.5 % thermophilic culture) --->incubate (40-45 degrees celsius, 3 to 6 hours) ---> mix and dispense ---> cool (0 to 4 degrees celsius) *Cup style* Milk (Standardize non fat, lowfat, whole; nonfat milk solids; stabilizers, other ingredients) ---> Yogurt mix (homogenize) ---> Heat treatment (85 degrees celsius for 30 minutes) ---> Cool (40 to 45 degrees celsius) --->Inoculate (2.0 to 2.5% thermophilic culture) ---> Dispense ---> Incubate (40 to 45 degrees celsius) ---> cool(0 to 4 degrees celsius) *organisms* Two organisms are used - Streptococcus thermophilus - Lactobacillus delbrueckii subsp. bulgaricus • Both organisms are needed to make yogurt • Many commercial products contain more than one strain of each organism • Traditional ratio of S. thermophilus to Lactobacillus delbrueckii subsp. bulgaricus is 1:1

bulk culture and direct to vat starter cultures (how they are prepared and how they are utilized)

Large scale producers do not want to be generating these large starter culture inoculums • Two main alternatives - Bulk cultures • Allows the producer to bypass the scale-up of inoculum (scale-up is performed by the supplier) • Bulk culture generation conditions are optimized for high cell number and viability in the resulting bulk culture • Producer can inoculate a bulk tank with a bulk culture, after sufficient time, the contents of the bulk tank is used to inoculate the production vat - Direct-to-vat cultures • Starter culture has been grown in large volumes followed by concentration - Results in very high cell densities • Use the direct-to-vat cultures to directly inoculate the production vat (do not need a bulk tank)

Steps for making cultured buttermilk

Milk (Standardize (nonfat,lowfat,whole); nonfat milk solids; stabilizers,other ingredients; citrate, salt) ---> Buttermilk mix (homogenize) ---> Heat treatment (85 degrees celsius for 30 minutes) ---> cool (20 to 22 degrees celsius) ---> Inoculate (0.5 % mesophilic culture containing acid producing and citrate fermenting strains) ---> Incubate (20 to 22 degrees celsius, 12 to 16 hours) ---> Cool and agitate (8 to 10 degrees celsius) ---> dispense ---> cool (4 degrees celsius) • Starter cultures - Acid producers • Homofermentative • Often Lactococcus lactis subsp. lactis or L. lactis subsp. cremoris - Flavor producers • Heterofermentative • Diacetyl producers • Often L. lactis subsp. lactis (diacetyl producer) or Lueconostoc mesenteroides subsp. cremoris • Have the ability to ferment citric acid to diacetyl

natural fermentation description

Relies on the indigenous microorganisms present in the raw material - Raw milk often harbors the bacteria necessary for fermentation - Very few commercial food products available in the US are produced using natural fermentations

Homofermentative vs Heterofermentative pathways

These are two pathways for sugar metabolism *Homofermentative LAB* • >90% of the sugar is converted to lactic acid • Uses the Embden-Meyerhoff pathway - Possess aldolase • Terminal step of fermentation is catalyzed by lactate dehydrogenase (LDH) - Converts pyruvate to lactic acid - Regenerates NAD • Produces two moles of ATP per mole of glucose *Heterofermentative LAB* • ~50% of the sugar is converted to lactic acid • Also make acetic acid, ethanol and carbon dioxide • Lack the enzyme aldolase - Does not use the Embden-Meyerhoff pathway • Uses the phosphoketolase pathway - Phosphoketolase is the key enzyme • Produce one mole of ATP per mole of glucose • More important than homofermentative LAB in production of flavor and aroma compounds • Product yields for both pathways may vary during actual fermentation - Type and concentration of substrate - Growth temperature - Atmospheric conditions - Growth phase of the cell • Example: homofermentative lactococci can divert some pyruvate away from lactate and towards alternative end products (acetate, carbon dioxide, etc.) when under sugar limitation conditions

backslopping

Transfer a portion of a successful fermentation to fresh raw material to initiate a new fermentation - Still used by some producers, especially small-scale and home production • For example, you can make yogurt at home by using commercial plain yogurt as a source of desired cultures

Lactic Acid Bacteria (LAB) Genera used in food fermentations and those that are not

Used in food/beverage fermentations- •Lactococcus* - cheese, cultured dairy products • Streptococcus* - cheese, yogurt • Lactobacillus* - cheese, yogurt, sourdough bread, sausage, • Leuconostoc* - cheese, cultured dairy products, fermented vegetables • Oenococcus* - wine • Pediococcus* -Sausage, fermented vegetables •Tetragenococcus* - soy sauce Not used in food/beverage fermentations - • Vagococcus • Weissela • Carnobacterium • Enterococcus • Aerococcus

starter cultures

Uses pure cultures of microorganism(s) to initiate fermentation Pasteur demonstrated that microorganisms were responsible for various food fermentations • Microbiologists searched for microorganisms that were responsible for various fermentations BULLSHIT ABOVE ^ • For many food and beverage fermentations, we can use pure cultures of microorganism(s) to initiate fermentation (starter cultures) Modern, large-scale production of fermented foods and beverages require: - Consistent product quality - Predictable production schedules - Stringent quality control to ensure food safety • The use of starter cultures has allowed largescale production facilities to meet these requirements For most fermented foods, the first requirement of a starter culture is that it initiate a fermentation promptly and rapidly • To do this, the starter culture must contain a large number of viable microorganisms Starter Culture Example • Typically, a 1% inoculum is used to ensure a rapid fermentation

Why do you need two different species of LAB to make yogurt?

• Both species are required to make yogurt with the desired properties • S. thermophilus and L. delbrueckii subsp. bulgaricus grow better together than separately

Milk composition

• Carbohydrate -rich and nutrient -dense composition - Composition of fresh cow's milk: • 5% lactose • 3.3% protein • pH 6.6 - 6.7 (ideal conditions for most microorganisms)

Basic functions of LAB (lactic acid bacteria) in dairy fermentations

• Ferment lactose to lactic acid - Decreases pH - Reach the isoelectric point of casein • Casein: major milk protein • Isoelectric point: pH at which the net electrical charge of a protein is zero and the protein is at its minimum solubility • For casein, isoelectric point is 4.6 • When sufficient acid has been produced to cause the pH to reach 4.6, casein precipitates and a coagulum is formed • The culture may also produce other small organic molecules (acetaldehyde, ethanol, diacetyl, acetic acid) - Relatively low amounts but important for the overall flavor profile of the product • The culture may also produce other compounds that contribute to the viscosity, body, and mouth feel of the product • The choice of LAB cultures is dictated by the product being produced

Kefir

• Fermented dairy product • Very popular product in Middle East, Eastern Europe and Central Asia - Accounts for as much as 70% of all cultured dairy products consumed worldwide • Drinkable yogurts are only slightly different than kefir

LAB and milk

• LAB are saccharolytic and fermentative • Saccharolytic: Capable of hydrolyzing or otherwise breaking down a sugar molecule. • Fermentative: Produce ATP via glycolysis; lack respiration - Ideally suited for milk - In general, LAB will out-compete other microorganisms for lactose - In addition, acidification will produce an inhospitable environment for would -be competitors

yogurt co culture dynamics

• S. thermophilus grows initially - Lowers pH to acceptable level for L. delbrueckii subsp. bulgaricus - S. thermophilus is weakly proteolytic and relies on the small pool of free amino acids in milk • S. thermophilus quickly uses up all of the available amino acids • L. delbrueckii subsp. bulgaricus produces a proteinase that provides amino acids for both organisms • Eventually, L. delbrueckii subsp. bulgaricus will produce more acid than can be tolerated by S. thermophilus - Prevents the continual co-culture of both organisms - To make yogurt starter cultures, the organisms are grown separately in species -specific optimum conditions, harvested and mixed in the desired ratio *fermentation conditions* • Incubated between 40°C - 45°C (thermophilic culture) - In general, L. delbrueckii subsp. bulgaricus has a higher temperature optima than S. thermophilus • < 42°C favors S. thermophilus • > 42°C favors L. delbrueckii subsp. bulgaricus • Incubation temperature influences the growth rates of the two organisms and the metabolic products they produce - For example, L. delbrueckii subsp. bulgaricus is capable of producing more acetaldehyde compared to S. thermophilus • Fermentation usually lasts 4-6 hours and is stopped when the pH reaches about 4.4 to 4.6 - Lactose concentration in yogurt is often higher than that of milk due to the addition of non-fat milk solids (often >6% lactose in yogurt)

compare and contrast cultured buttermilk and sour cream manufacture

• Very similar to cultured buttermilk - Notable exceptions: • Cream instead of milk • Milder heat treatment

steps for making sour cream

• Very similar to cultured buttermilk - Notable exceptions: • Cream instead of milk • Milder heat treatment Cream (standardize to 18 to 20% (less for reduced fat versions), stabilizers and other ingredients) ---> Sour cream mix (homogenize) ---> heat treatment (85 deg celsius for 25 seconds) ---> cool (20 to 22 degrees celsius) ---> inoculate (1% mesophilic culture) ---> incubate (20 to 22 degrees celsius, 12 to 16 hours) ---> cool and dispense (8 to 10 degrees celsius) ---> cool (0 to 4 degrees celsius)