SUGGESTED QUESTIONS FOR FINAL EXAMINATION REVIEW 

ON ENZYME AND FOOD FERMENTATION   

Question 1: What do you know about the starter culture. Clarify its roles in food
fermentation.  

- Starter culture is the preparation of microorganism, initiating in fermentation

process. Starter culture contains microorganisms from natural microflora (lactic
acid bacteria, yeasts, molds, etc.) and are defined as mesophilic (e.g.:
Lactococcus lactis subsp.  cremoris) or thermophilic (e.g.: L.casei, L.plantarum).
- Generally, starter culture consists of microorganisms inoculated into food
materials, provides characteristics in a more controlled and predictable way
including enhancing preservation, improving nutritional value, sensory qualities and
economical values.
- Starter cultures take part in fermentation with some functions as mentioned
below:
● flavour, aroma, and alcohol production
Example: the performance of starter culture is crucial in industrial cheese making
as it affects cheese flavor. By controlling acidity, the over curd pH, moisture and
lactose level can also be controlled as these factors have impacts on maturing
cheese and coagulation.
● proteolytic and lipolytic activities:
Example: starter cultures will hydrolyse casein to various degrees based on
proteolytic activity of the starter strain. These strains rely on the rate of peptide
growth as well as pH level in cheese manufacture which can slow-down the
proteolysis and flavor development during maturation.
● inhibition of undesirable organisms
Example: the antimicrobial properties in lactic acid bacteria can reduce the growth
rate of undesirable microorganisms. LAB produce organic acids (acetic, lactic acid)
which inhibit food spoilage by against the gram-positive and gram-negative as well
as yeasts.
Question 2: Students choose one from two production technologies and by your best
understanding, discuss it, (1) Modern fermentation technology of beer product, (2)
Traditional fermentation technology of soy sauce product.

(1)
 ● Malting (including 3 sub-steps: steeping, germination, kilning)

Barley is harvested and cleaned for beer production. Firstly, they go through steeping
in which the grains are soaked with water for about 2 days. The germination ensues the
water uptake of barley grains. During germination, the grains are modified and start to
produce enzymes such as amylases and proteases. After 3-5 days, the germinated
malts are dried in the kilning stage for 1-2 days. Particularly, heat is applied to stop the
germination and also the activity of enzymes in the malt. Flavour and colour are also
developed during kilning.

● Mashing

The malts from the previous step are crushed in the mill to create grist. This grist then
enter the mashing stage for about 1-2 hours where it is mixed with warm water. The
temperature in the mixer is changed continuously for different enzymes to work.

✔ Amyglucosidase: increase the amount of glucose in wort

✔ β-Glucanases: break down cell wall in the malt which helps improve malting
and lower the viscosity of the wort
✔ Proteases: break down proteins and provide the beer with amino acids
which can be later used by the yeast. The degradation of proteins also
enhances the head retention of beer and prevent haze formation.
✔ Amylases: The activity of amylases is the most important to beer production.
Most of the time, the temperature of the wort is kept at around 65 oC where
the β-amylase work best. β-amylase has optimum temperature ranged from
60-65oC. β-amylase breaks down starch to produce maltose and glucose –
the primary sugars used by the yeast in the fermentation stage. On the other
hand, α-amylase works best at 70-75oC. The activity of α-amylase result in
the release of dextrins which are easier to break down by β-amylase due to
its simpler structure and water-soluble property.
 ● Boiling (brewing)

The wort is filtered, diluted and boiled for about 2 hours. Hops are added at this stage
and the boiling help extract hops flavour components. High temperature also
inactivates enzymes working in the previous stage. Aromatic and coloring compounds
are also formed during this stage. Boiling also helps sterilize and concentrate the wort.
Coagulation of proteins are also seen during this stage. After boiling, the wort goes
through the whirlpool separator to filter out the precipitated proteins and insoluble
components from the hot wort.

● Cooling

The wort is cooled to around 9oC and aired in sterile condition.

● Fermentation

The wort and yeast are pumped into the fermentation vessel. The wort is converted by
the yeast and ethanol and CO2 are released. After fermentation, green beer is produced
and excess yeasts are removed and saved for subsequent use. Cylindroconical tanks
(CCTs) are often used as the vessels for beer fermentation. Two main types of yeast
used for beer fermentation are ale yeast and lager yeast.

*Difference between ale and lager yeast (Phần này thấy thầy có nhấn mạnh nên tui bỏ
vô)

Ale Yeast Lager yeast

Saccharomyces cerevisiae (ale type) Saccharomyces carlsbergensis

Typical fermentation temperature 18- Typical fermentation temperature 8-15°C

25°C

Maximum growth temperature 37°C or Maximum growth temperature 34°C

higher

Does not ferment melibiose Ferments melibiose

Top fermenting yeast Bottom fermenting yeast

● Maturation
The beer is allowed to rest at suitable temperature. Taste and CO2 components
are adjusted and sedimentation of yeast and cold trub presents. This stage is
considered the secondary fermentation stage. CCTs are also used at this stage.

● Clarification

Yeast and cold trub are filtered and removed from the beer.

● Stabilization

The killing and removal of microorganisms by biologically stabilizing method. This

operation can be carried out coldly (sterile filtration) or hotly (pasteurization).

● Packaging

The filling of beer into bottles, cans, casks or kegs.

❖ Batch Fermentation Technology

The use of cylindroconical tanks (CCTs) for both fermentation and maturation are
considered batch technology which is widely used in modern breweries. These
vessels have a wide range of beneficial features:

⮚ They occupy a small land area in relation to volume;

⮚ They can be used for all beer types;

⮚ An increased dispersal of yeast by carbon dioxide uplift allows rapid

fermentation
⮚ Ease of pressure application, carbon dioxide collection, yeast and cold

break removal and cleaning (using a CIP system);

⮚ Efficient cooling; and

⮚ Simplified automation.

The vessels have high height-to-diameter ratios which result in an increase in

hydrostatic pressure. This increases the amino acids utilization by the yeast due
to increased fluid flow circulation via carbon dioxide evolution. As a result, the
beer will have higher alcohol and lower ester content. The temperature is closely
regulated using temperature sensor and cooling jackets (or pockets). In some
cases, primary fermentation and maturation can be performed in the same
vessel. This is called single-vessel processing.

Question 3: Yogurt and other fermented milk products are good for human health, choose one
product, and focus on the production technology and discuss it.

Yogurt making process brings many benefits to human health that should be taken into
consideration.

Milk reception: In milk reception, the quality of milk is really important. Chosen milk must
come from healthy cows that have low acidity and do not contain antibiotics residues.

Fat standardization: is the reduction in the fat content to 3.25%, the purpose is to create the
viscosity in milk and to maintain the adequate fat content in a food product. Fat is removed and
the addition of MSNF or skimmed milk occurred.

Homogenization (100-200atm, 50-60ºC): Milk is the oil-in-water emulsion that the fat globules
need to be adjusted to smaller sizes, or else there will be a layer of fat on the surface of milk if
the solution is being left under the atmosphere for too long.

Pasteurization(90-95ºC, 5-10min): kill the vegetative cells, inhibit the growth of microbes.

Cooling to 43ºC: at this process, starter culture is added for fermentation.

 Yogurt culture
The bacterial culture using for milk is called Lactic Acid Bacteria since these bacteria are used to
convert sugar to lactic acid. Streptococcus thermophiles and Lactobacillus is added under the
ratio 1:1 and under 43ºC. S.thermophile reduces pH in the culture, enhance the coagulation of
milk, produce organic acids. Lactobacillus breaks down milk proteins to small chains of amino
acids. These amino acids create the aroma in yogurt.

Lactose -------> glucose -------> lactate

Milk protein ---------------> Peptide ------------> amino acid -> sour flavor in yogurt.

Functions of starter culture: the natural microflora of the milk is either inefficient,
uncontrollable, and unpredictable, or is destroyed altogether by the heat treatments given to the
milk. A starter culture can provide a particular characteristic in a more controlled and predictable
fermentation.

The primary function of lactic starters is the production of lactic acid from lactose.

Other functions of starter cultures may include the following:

Flavor, aroma

Proteolytic and lipolytic activities

Inhibition of undesirable organisms

After the fermentation process, yogurt will be cooled down to the packaging.

Packaging: After packaging and overpackaging, the yogurts are stored at low temperature (4 or
5oC)

Limiting the post-acidification in the products

Preserving their safety towards the contamination from the environment

Question 4: What do you know about fermented foods and human history.

Fermentation is one of the oldest biotechnologies for the production of food products with
desirable properties such as extended shelf life and good organoleptic properties.
The origins of fermentation are hard to track down along with human history. However,
historians have traced signs of fermentation in food and beverage preparation dating as far back
as 7000 BC. Asian civilizations, in particular, have a history of fermenting a wide variety of
foods—Japanese natto (soybeans), Vietnamese mắm (seafood), Chinese douchi (black beans)
Korean banchan (side dishes)—that remain essential components of their everyday cuisine.

Early humans began to embrace this partnership with microbes because of its benefits, namely:
more digestible food, food that kept longer, was less likely to make you sick or simply tasted
better than unfermented foods. During winter, when our ancestors cannot go outside and hunted
for raw material, the remaining food materials were used to get through the toughness time of the
year, however, it easy to be spoiled and inability to consume. That is the reason why fermented
food created to prolong the food shelf-life as well as giving a flavorful taste that contribute to
flavor diversity.
Besides, vegetable and fruit, dairy- which have significant water content, are prone to damage.
Hence, for longer preservation time, fermented vegetable such as kimchi, sauerkraut, pickles
were born along with wine from grape fruit and cheese curd formation from animal milk.
The initial mechanism for fermentation during this time was wide yeast or LAB bacteria that
already exist in the food materials as well as temperature.
However, unexpected phenomena could happen such as the growth of undesirable microbes
which can lead to food borne illnesses, and even death during that time.

French chemist Louis Pasteur figured out the way to minimize the negative effect of unwanted
microbes in fermentation, his pioneering work uncovering the role microbes play in fermentation
as well as less desired outcomes like sickness and food spoilage. His invention, pasteurization is
still used today to kill microbes that could cause sickness or cause food and drink to spoil. 
Pasteur's finding showed that there are two types of fermentation: alcoholic and lactic acid.
Alcoholic fermentation occurs by the action of yeast; lactic acid fermentation, by the action of
bacteria.

Modern large scale production of fermented foods and beverages is dependant almost entirely on
the use of defined strain starters, which have replaced the undefined strain mixture traditionally
used for the manufacture of these products.

Today this ancient knowledge has been combined with basic scientific knowledge and applied
toward modern production processes. These industries are the result of the laborious work of
hundreds of scientists who were curious about how things work.

Question 5:  Discuss about roles of fungi, bacteria and yeast in food processing

- Fungi, bacteria and yeast can be used in fermentation of food.

- Fungi, especially Penicillium, Aspergillus fungi, play a key role in food fermentation:
both are used in cheese making as they produce microbial rennin which can
change the flavour of cheese.
- Bacteria is used in a wide range of food products. Lactobacillius species or lactic
acid bacteria is the most common bacteria used in food products and play a
crucial role in almost food fermentation processes such as brewing, baking, cheese
and butter making.
- Yeast is unicellular of fungi, but larger than bacteria. Yeast strains are also used in
lactic acid fermentation of fruits to produce wines, cereals to make beer and
commonly used as a leavening agent in the production of bread by breaking down
sugars into alcohol and co2. Alcohol gives alcoholic beverages (beer, champagne)
their bubbles and improve the bread dough.
Question 6: What do you know about cheese fermentation

After the process of milk choosing, fat standardizing and pasteurization. Milk will be
cooled down to room temperature to add the starter culture for fermentation.

Inoculation:

By adding the culture, cheese will have a lower pH, which initiates fermentation.
Depending on the type of cheese, different types of cultures are preferred.

In inoculation, main LAB is added to enhance the fermentation process. Secondary

culture is lactic culture adjuncts, molds.

For example, the making of Limburger cheese will be added with tortula yeast.

The culture for blue cheese is Penicillium Roqueforti.

Coagulation:
After Lactic acid is produced, rennet is added to help to coagulate the milk protein.
Rennet contains chymosin, an enzyme derived from an animal’s intestine, which
hardens the milk and converts k-casein to para-k-casein.
This process changes the acidity level of the milk and starts the process of thickening
the milk into a solid. Next, rennet is added to encourage even more thickening so that
curds form and separate from the whey (liquid).
After coagulation, there will be two parts for cheese: curd and whey, whey will be
removed for different purposes, curd will be kept to continue the process.

Draining:
The curd is separated from the whey by draining. The curd can be used directly to make
unripened cheeses such as ricotta or cottage cheese or can undergo further processing
to make a ripened cheese.

Ripening:
The ripening process requires addition of salt, 0.65% salt is added. Dry salt is added on
curd or placed in brine (salt solution).
Example: During this time, enzymes and bacteria continue to modify proteins, fats and
sugars in the cheese. The holes in Swiss cheese occur during ripening -- Swiss cheese
is ripened in a cool place for several weeks, then put in a warm place (70 degrees F, 21
degrees C or so) for four to six weeks, where special bacteria ferment the remaining
lactose and produce carbon dioxide bubbles in the cheese.
7.Discuss meat fermentation
Fresh meat is a nutrient-rich medium that consists of 20 -25 % protein, 75% water,
2-3% of lipid, and a small amount of carbohydrate. Since rich nutrient and ideal pH level
of 5.6-5.8 after rigor mortis can favor the growth of spoilage microorganisms and
pathogens, fermentation is applied as a preservation method results in meat
products with longer shelf-life and improvements in color and taste.

Input for fermentation can vary from the meat source (i.e., beef, pork, goat, sheep,
etc.), but the cut of meat, the amount of the fat, and the casing material. Fermented
meat products such as salami, ham, sausage vary in texture, flavor, safety.

fermentation: meat after preparing was added with starter culture and then sent to
fermentation. After being fermented, meat products would go through other processing
steps before being packaged.
● cutting: fresh meat after being collected is washed and removed the inedible
part. Then it is cut into pieces
● addition of curing mixture and culture:
curing mixture: salt, sugar, nitrite (nitrate), reducing agent, antioxidants
Salt solubilizes the meat protein, increases the osmotic pressure which
suppresses spoilage pathogens
sodium nitrite promotes the typical color of meats (formation of nitric oxide
compounds by reaction with the haem of myoglobin) inhibits the development of
pathogens Clostridium botulinum.
The disadvantage is the production of the potentially carcinogenic nitrosamines
which require legal limits on dose of use. Nitrate may replace nitrite, in which
case it is converted to nitrite through the action of bacteria.

Sugar balances the salt flavor, the carbon, and energy source for
fermentation, Maillard reaction to impact color and flavor.
Binding agents and emulsifiers may be used to improve stability.
antioxidants counter the development of rancidity through lipid oxidation.
culture:
lactic acid bacteria were the primary organisms responsible for the fermentation.
dominant organisms isolated from naturally fermented sausages were species of
Lactobacillus.

traditional fermentation: back slopping

addition of a part of the finished product from the previous batch. effective due
simply to the size of the inoculum, however, products often have inconsistent
quality and fermentation can be unreliable and difficult to control
industrial fermentation:
 Starter cultures are primarily the lactic acid bacteria lactobacilli and pediococci,
such as Lactobacillus sakei, Pediococcus pentosaceus, Lactobacillus curvatus,
Lactobacillus plantarum and Lactobacillus pentosus

● Fermentation: During fermentation, LAB will convert glucose and other sugar into
lactate (lactic acid in solution). The acid produced in fermentation promotes
the reaction of nitrite with metmyoglobin to produce NO-myoglobin. Low pH 4-4.5
and anaerobic condition are achieved for the fermentation. Temp is controlled at 22-
26 with a minimum value at 20C. lower incubation temperatures require longer
fermentation times. the access of oxygen to the meat will be minimized at all stages

The flavor and aroma that develops are due to the degradation of lipids, notably
through autoxidation and the microbial transformation of the products generated by
lipid degradation. proteinases produce peptides that are converted by the
microflora to amino acids and volatile fatty acids.
The low acidity of the fermented product prevents the growth of spoilage
microorganisms and pathogens.
● Further processing
Several different treatments and combinations of treatments can be applied at the end of
the fermentation. These include cooking, drying, and smoking.
Drying and smoking not only aid in flavor and color to the fermented product but also
help in achieving the desired aw that plays as hurdles effect in increasing the shelf-life
and safety of products against pathogens.

8.Sauerkraut is a sour-flavor fermented food made from cabbages with salt,

and it is probably the most well-known lacto-fermented vegetable along with
Kimchi- traditional side dish of Korea.

Processing:
A tightly compacted cabbage, larger cabbages tend to be milder in flavor than
smaller ones. Remove the outer leaves as well as the core of cabbage and then
clean the cabbage from contaminants under running tap.
Cut or shred cabbage into smaller particles for best texture while consuming.
Put the sliced/shredded cabbage into the big mixing bowl (or tank in industry) and
then add specific amount of salt (2%- 3% of salt)
Knead and squish the mixture of cabbage and salt until the brine is achieved
enough amount of liquid. Transfer treated cabbage into container and then press in
tightly. By pressing the cabbage, we can get rid of remaining oxygen content in the
air and create a good condition for fermenting anaerobes to work.
Mechanism: At a certain salt concentration, lactic acid bacteria grow more quickly
than other microbes, and have a competitive advantage. The salt along with lack of
oxygen condition in container will allow the optimal range of LAB to work. LAB
release the lactase dehydrogenase to catalyze the reaction corresponds to the
formation of lactate (lactic acid bacteria) Homofermentative and
Heterofermentative both occur.

Carbohydrate =Glycolysis=> Pyruvate=Lactase dehydrogenase=> Lactic acid =>

The sour taste of fermented vegetable.

Pour the juice of mixture into the jar and make sure the water/brine covers the
cabbage entirely. Weight is required to submerge the cabbage down below the
liquid.. Cover it carefully for giving optimal condition (oxygen content) for LAB
to work Allow the cabbage to ferment at room temperature and away from direct
sunlight. . Fermentation will begin within a day and take 2-5 weeks depending on
temperature.

In industrial process, the additional pasteurization is required before being

packaged; however, both good and bad bacteria are inactivated during this step

Advantages:

• Boost immune and cognitive system

• Provide both prebiotic and probiotic for digestive system

• Better in weight loss

• Strengthen your bones

Disadvantages:

● Excessive consumption will lead to bloating and flatulence

● People with an unhealthy gut cannot handle the acidity of an excess amounts
of sauerkraut, otherwise, inflammatory bowel disease may be worse.
● Unwell-treated sauerkraut can result in mold growth and other contaminants.
9. discuss the production of sausage
Sausage is one of the oldest known forms of processed meat. It is considered one of the most
appetizing, nutritious, enjoyable, and convenient meat products.

Sausage is ground or chopped meat combined with salt, seasonings, and other ingredients, which
can be stuffed into a container or casing of particular shape and size

Sausage making is a traditional food preservation technique. Sausages are classified via their types,
based upon processing procedures and product characteristics. Fresh sausage, dried sausage,
fermented sausage, smoked sausage

Ingredients:
Meat: Sausage making is a great way to use less tender, low-value cuts and trim pieces. Beef,
veal, pork, lamb, mutton, and poultry are all suitable for use in sausage

salt: It aids in the water binding and emulsifying capacity of meat proteins. necessary for
enhancing flavor, preserving the sausage from microbial spoilage, and extracting the soluble
meat proteins. The extracted meat protein forms a film and coagulates during heating and binds the
meat particles together providing a firmer texture for sausage.

sugar flavor development and to counteract the slight, bitter taste of salt. used as a substrate for
microbial fermentation to reduce the pH of the dry and fermented sausage.

spice found in many forms: fresh, dried, whole, crushed, pureed, as paste, and as an extract.

nitrates and nitrites one of the forms of food preservation. imprové food safety with extended
shelf life and excellent storage stability. imparts unique colors, texture, and flavors

Nitrate and nitrite are converted to nitric oxide by microorganisms and combine with the meat
pigment (myoglobin) to give the characteristic pink cured-meat color. provides protection against the
growth of botulism-producing microorganisms, acts to retard rancidity, and stabilizes the flavor of
cured meat

starter culture an inoculum of bacteria that produce lactic acid from sugar.

antioxidant Ascorbates and erythorbates are active reducing agents that react with nitrite to
accelerate the curing process

reducing agent retard oxidative rancidity and protect flavor

binders and extenders retain natural juices and reduce the cost of the formulation. y improve the
binding characteristics, flavor, cook yield, slicing characteristics, and reduce the cost of the
formulation

water added to most sausage formulations to rehydrate the nonfat dry milk and to replace the
expected moisture loss during smoking and cooking.
casing most sausages are either wrapped or packaged in bulk chub packs or stuffed into casings.
Two types of sausage casings are natural and synthetic. Natural casings are obtained from the
digestive tract of animals. Edible synthetic casings made from collagen are also available, in
approximately the same sizes as the natural casings

Procedure of making sausage

1. mixing meat are cut into desirable sizes. spices and dry ingredient are added and mixed
thoroughly. water, ice, or wet ingredients last and mix again.
mixing will ensure a uniform distribution of spices and develop the binding ability of the meat.
2. Grinding small manual and power grinders is needed to grind all the mixed ingredient
together. the size and coarseness of particle depends on specific type of sausages
3. Stuffing and lingking: the blended ingredent nay be bulk packed or may be extruded into
casing
4. Further processing: stuffed sausages can go through further processing unit. Sausauge
can be cooked and consumed immediately, however, they need to be store refrigerated or
freezed.
Sausage can be smoked or dried as a cooking step. This lengthen the shelf-life and achieve
desirable aw. Smoking also imparts flavor and aroma and inhibit the growth of pathogens.
There are different smoking method: hot smoking, cold smoking, liquid smoke. Smoking can
be obtained from burning wood or using smoke generator
5. packaging: finished sausages is packaged before reaching to the consumer. Specific
packaging vary depending on the types of sausage, the needs of consumer, hygiene
standard in processing. Vacuum package, plastic wrap, zipper bag can be used

Important parameter in sausage production

Temperature Meat products are extremely perishable and must be maintained under
refrigeration (40º F or below).
The temperature of drying, smoking, and cooking must be continuously and carefully
monitored and controlled to ensure a consistent product. Processing temperatures and times
must be sufficient to eliminate any pathogens of concern.

Fat content: Different sausages have different amounts of fat. Avoid making the formula too
lean as the sausage will be too dry and hard. Fresh pork sausage contains 30 to 45 percent
fat. Smoked or roasted sausage contains 20 to 30 percent fat

Sanitation: Cleaning and sanitizing are among the most important activities in meat product
plants, Sanitation and proper handling of meat and meat products are very important in
sausage making.
Bacterial contamination and foodborne illness are the main food safety concerns during raw
meat handling and sausage making. In order to prevent the spread of pathogenic bacteria,
the food preparation area and equipment must always be cleaned and sanitized before and
after processing sausages.

Question 10: What do you know about the traditional fermentation of Chao, a

traditional food of Vietnamese
Chao is basically fermented tofu, a very popular local Vietnamese food. Chao has a
special fatty and sweet flavour which resembles the Roquefort blue cheese. There are
two types of Chaos: dry and wet Chao. The wet Chao is usually added with salt water
and rice alcohol after 2-3 days of fermentation whereas the dry Chao is kept away from
any water sources.

Process involved in fermenting Chao:

● Boiling: The tofu is boiled in salt water for a couple of minutes. This process
helps the tofu to drain more water and kills some bacteria simultaneously
without damaging the mold culture.
● Pressing: The tofu is covered in paper towels and pressed by a weight for about
1-2 hours. This pressing will remove excess moisture within the tofu.
● Cutting: The large tofu is cut down into small pieces (usually into small cubes)
so the heat can be evenly applied to each portion of the tofu.
● Primary fermentation: Tofu cubes are covered in paper towels and allowed to
ferment for about 2-3 days at 25-30 oC. The fermentation imparts a light-yellow
color and unique smell on the tofu as a result of the proteolytic activity of mold
enzymes. The tofu now attains the desired culture and mold quantity for
secondary fermentation.
● Brining: A brine is prepared to prevent the Chao from getting spoiled and also
enhance the flavor. Basically, the brine contains boiled water mixed with salt,
sugar, and alcohol (usually rice whine or vodka). The Chaos are put in a jar filled
with brine and then sealed to maintain the culture.
● Secondary fermentation: The jars of chao are placed in a dark place at around
20oC. This process is also considered as aging process. The aging time
depends on the desired texture and flavour of the Chao. The longer the Chao is
allowed to age, the creamier texture and more intense flavor it could attain due
to molds activity. Generally, Chao is often fermented for at least 3 weeks.