Fermentation Processes Quiz

Agricultural Microbiology - MICR20010

• Course code is MICR20010
• Course title is Agricultural Microbiology
• Instructor is Dr. Tadhg Ó Cróinín
• Practical MCQ exam results will be released this evening.
• Exam results are output as a percentage then converted to a letter grade using an alternative linear scale
• A+ is 95% and above to 100%
• The alternative linear scale is provided with the percentage ranges for each grade

Remaining Lectures

• Lecture 10: Microorganisms and Disease
• Lecture 11: The Immune System
• Lecture 12: Pathogenic Bacteria
• Lecture 13: Pathogenic Fungi and Viruses
• Lecture 14: Antibiotic Resistant Microorganisms
• Lecture 15: Microbiology in the Food Industry - The Fungi
• Lecture 16: Microbiology in the Food Industry - Fermentations
• Lecture 17: The Nitrogen Cycle

Fermentations - Food and Drink

• An ancient process that existed before the study of microbiology.
• Critical for the production of Beer, Wine, and various Dairy products

Fermentation Pathways

• Lactobacillus:
  • Glucose → fructose diphosphate → 2 phosphoglyceric acid →2 pyruvic acid → 2 lactic acid
• Saccharomyces:
  • Glucose → fructose diphosphate → 2 triose phosphate →2 pyruvic acid → alcohol + CO₂

Fermented Vegetables

• Soya bean fermentation

  • Koji: Aerobic fermentation with Aspergillus
  • Moromi: Anaerobic fermentation with Tetragenococcus halophila

• Sauerkraut

  • Lactobacilli fermentation of shredded cabbage
  • Salt prevents Gram-negative contaminants

Fermented Meat and Fish

• Various dry sausages (e.g., salami, pepperoni, bologna) use Staphylococcus, Pediococcus, Micrococcus, and Lactobacilli.
• Fish sauces are a variety of sauces and pastes in Asian cooking, sometimes using fish and shrimps, and salt, in sealed vessels to allow natural microflora (like S. carnosus and Staphylococcus piscifermentans) to work

Coffee and Cocoa

• Coffee (Coffea arabica):
  • Wet or dry process to obtain beans from berries
  • Wet process uses indigenous fungi and bacteria, secreting proteolytic enzymes, followed by lactic acid bacterial fermentation.
• Cocoa (Theobroma cacao):
  • Pods are opened to remove beans.
  • A sequence of fermentations (involving yeasts, lactic acid and acetic acid bacteria) occurs before the beans are processed further.

Brewing

• Ancient use of fermentation, a very important economic activity
• Primarily Saccharomyces cerevisiae but occasionally Zymomonas.
• Zymomonas produces African palm wine or Mexican pulque

The Basics (Brewing)

• Aerobic pathway is preferred by yeast, so need to reduce oxygen to promote fermentation and alcohol production.
• CO₂ produced must be allowed to escape.
• Glucose as a substrate provides the starting material

Winemaking

• Grapes are the source of sugar
• Fructose + Glucose are used as sugars, and natural acidity is important
• Soil, climate, grape variety, pressing method (primary and secondary fermentations) influence wine characteristics.
• Red wine is made with skins containing anthocyanins

Making the "Must"

• Grapes are crushed mechanically or by hand (feet)
• Fermentation is driven by indigenous yeasts or starter cultures.
• Fermentation rate is impacted by temperature, pH, initial sugar levels, and yeast strain

The Fermentor

• Typically wooden fermentors, but currently mostly stainless steel.
• Cooling is necessary to manage heat produced (1.3°C per 10g/L)
• Unwanted yeasts are controlled by SO₂
• Starter culture of S. cerevesiae added
• Secondary fermentation may occur
• Clarifying agents and filtration are used, followed by bottling

Cider

• Cider production is similar to winemaking
• Additional sugar may be added to help the reaction
• Apple juice already has a high sugar content

The Problem with Barley

• Barley contains 65% starch as a sugar source
• Starch needs to be broken down into simpler sugars for easier utilization

Malting Process - Partial Germination

• Enzymes are generated (amylases) and substrates for fermentation are created during the malting of the grain.
• Green malt, created after five days of germination, undergoes kiln drying and partial cooking using hot air.
• Malt is preserved using kilning

Making Malt - The Steps

• Soak or steep barley for 2 days at 10-16°C.
• Occasionally aerate.
• Germinate for 3-5 days at 16-19°C on malting floors, a process that requires aeration and mechanical turning.
• Subsequently kiln dry by heating to 50-60°C and then 80-110°C to stop enzyme development, prevent enzyme denaturation, and add flavour

Making Wort

• Malted grains supply both enzymes and substrate for fermentation, but additional adjuncts are useful.
• Starch conversion into simple sugars
• Wort is prepared (mashing) as a glucose source.

Mashing - Making the Wort

• Allows endogenous and added substrates to be acted upon by enzymes.
• Various types of mashing are used, such as decoction mashing.
• In decoction mashing, the process begins at 35-40°C, with some of the mash being boiled, and reintroduced to incrementally increase the mash temp (and sometimes repeated) for protein rest
• In infusion mashing, a specific temperature is maintained throughout the process

But What is Happening?

• Amylase and other enzymes break starch down into fermentable sugars.
• Proteases also break down cell walls to enable starch access.
• If enzyme activity is limiting, commercial enzymes can be added.
• The sweet wort is then boiled.

Wort is Boiled

• Amylases are inactivated.
• Starch breakdown is stopped.
• Alpha acids and oils from hops to kill microbes and give flavour to the wort
• Sugars are caramelized

Hops

• Bitter herb grown on a vine
• Stabilizes flavour as well as providing preservative in brewing.
• Hop oils contain alpha acids for a bitter taste and a preservative element

Brewing (Wort Addition)

• Wort is prepared, and yeast is added
• Fermentation is allowed to occur for approximately 3 days
• Afterwards, a further 10 days are allowed for fermentation in the production of beer

Brewing: Types of Brewing Yeasts

• Top fermenting yeasts
  • Remain distributed throughout the wort.
  • They are carried up to the top by CO₂ production.
  • Ales utilize 14-23°C fermentation temperatures.
  • S. cerevisiae is commonly used.
• Bottom fermenting yeasts
  • Settle to the bottom.
  • 6-12°C fermentation temperatures are used.
  • Saccharomyces carlsbergensis is commonly used (a larger yeast compared to other yeasts.)

Differentiation of an ale yeast from a lager yeast

• S. cerevisiae and S. carlsbergensis can both grow at 27°C, but S. cerevisiae can grow at 37°C. S. carlsbergensis cannot grow at 37°C

Brewing (Post-Fermentation)

• Allow yeast to settle at the bottom
• Siphon off beer into bottles, allow to age further.

Distilled Beverages

• Alcohol boils at 78°C
• Malt (e.g., Whiskey)
• Wine (e.g., Brandy)
• Molasses (e.g., Rum)
• Grains/potatoes (e.g., Vodka)
• Colour is affected by barrel aging

Economic Importance

• Largest consumer of alcohol in Europe.
• Brew history in the region.
• Local breweries are significant economic contributors

Scaling up Ethanol production

• 50 Billion liters are produced from the fermentation of various feedstocks.
• Ethanol serves as an industrial solvent and a biofuel.

Vinegar

• Not only a condiment, but also an important preservative.
• Solution containing greater than 4% acetic acid.
• Different sources (e.g., America-cider, Europe-Wine, Britain-Malt) utilize different production approaches focusing on different substrates.
• Acetic acid bacteria are crucial for vinegar production.

Vinegar Production

• Changing ethanol to vinegar involves the conversion of ethanol to acetic acid using acetic acid bacteria.
• Distilled vinegars are commonly produced by this method.
• Production involves a recirculation tube, pump, wood grating, collecting chamber, beechwood shavings, oxidation air intakes, and cooling coils.

Fermented Dairy products

• Milk is 87% water
• Milk proteins: whey and casein.
• Fat-flavor components.
• Carbohydrates: lactose.
• Vitamins

Microorganisms Involved in Dairy Fermentations

• Lactococci, Lactobacilli, and S. thermophilus.
• Can be single or mixed cultures.
• Other microorganisms can be used to influence flavor and texture

Curdling Milk

• Acidification of milk coagulates milk proteins (curd).
• This can be achieved through a pure starter culture or a mixed culture.
• Rennet, an enzyme mix that promotes curdling (traditionally calf chymosin, now often fungal proteases).

Dairy Products (Cheese Production)

• The production process involves pasteurization, standardization, and filtration.
• Rennet is used for curdling.
• Curds are drained of whey and poured into molds.
• Salting, pressing, and ripening processes occur.

A Second Inoculum

• After salting the curd, a second microbial inoculum is added.
• Different inoculants:
  • Swiss cheese - Propionibacterium freundenreichii (flavor, gas bubbles)
  • Blue cheeses - Penicillium roqueforti (inoculated into cheese)
  • Camembert - Penicillium camemberti (characteristic rind and flavor).

Huge Variation in Cheeses

• A wide variety of cheeses exist due to differences in the production processes

Dairy Products (Other)

• Other types of fermented milk products include yogurt, buttermilk, and sour cream.
• Streptococcus thermophilus is important in yogurt production.

Yogurt

• Controlled fermentation of milk is essential.
• Lactobacillus bulgaricus and Streptococcus thermophilus are used for production.
• Pasteurization, addition of Streptocuccus thermophilus, Lactobacillus bulgaricus, incubation (at 42-45 C) generates yogurt.
• Symbiotic event leads to protein breakdown and release of peptides by L. bulgaricus, acid production (folic and formic acid) used by L. bulgaricus to synthesize purines by S. thermophilus

The Probiotic World

• "Good bacteria" theory.
• Elie Metchnikoff (1845-1916) began the study.
• Pasteur institute in France created "la Ferment”.
• Minoru Shirota (1930s) isolated L. casei Shirota from human feces.
• Yakult now produces these probiotics.

The Probiotic World (Additional Notes)

• Bifidobacteria and lactic acid bacteria are commensals in the gut (16 and 3% of normal flora).
• They are thought to be effective in treating antibiotic-induced diarrhea, IBS, IBD, and possibly neurological conditions.
• More study is needed to definitively prove probiotic benefits

In Summary

• Fermentations are crucial in industrial microbiology.
• They are critical in the dairy and beverage industries, particularly in cheese, yogurt, probiotic, beer, and wine production.
• S. cerevesiae is important for beer production.
• Acetic acid bacteria are essential for vinegar production.

Final Lecture

• Final lecture on Friday, covering the Nitrogen Cycle and Environmental Microbiology.
• A study guide will be provided on how to prepare for the exam.

Test your knowledge on the various fermentation processes used in food production, including coffee, cocoa, wine, beer, and yoghurt. This quiz covers the role of specific bacteria and yeasts, as well as critical steps in each fermentation process. Perfect for students of food science and microbiology.