Vegetable fermentation

Question 1

What are the primary fermented vegetables in Canada?

Answer 1

• Cucumber Pickles (can also be acidified, pasteurized, and refrigerated)
• Olives
• Sauerkraut

Question 2

Make a table of five products from Germany, Korea,Vietnam (Dhamouoi), Thailand (Dakguadong), and Philippines (Burong mustasa), major ingredients, microorganisms

Answer 2

Question 3

What is the principle of fermentation and preservation of fermented vegetables?

Answer 3

• Prior to fermentation, fresh fruit and vegetables harbor a variety of microorganisms included aerobic spoilage microflora, as well as yeasts and molds
• The cell populations on raw vegetables range 104 to 106 CFU/g
• Brining the vegetables (adding salt) results in the production of organic acids, and a variety of antimicrobial compounds by LAB
• LAB are initially present on fresh vegetables in lower numbers (102 to 103 CFU/g) compared to other mesophilic organisms
• During the fermentation, organic acids diffuse into the brine, and the low pH that results, influences microbial growth in the vegetable material
• As the vegetable cells die, sugars diffuse into the brine resulting in the rapid growth of LAB
• In the absence of brine, spoilage microbiota are able to grow

Spoilage bacteria result in the deterioration of the vegetable material due to the elaborate destructive enzymes produced (proteases, lipases, amylases, nucleases, etc

Question 4

What microorganism is important in initiating fermentation

Answer 4

Leuconostoc mesenteroides

Question 5

What are growing conditions for L.mesenteroides, at what acidity it will not grow, how it favors the preservation of cabbage

Answer 5

• Leuconostoc mesenteroides are important in the initiation of the fermentation of several vegetables
• L. mesenteroides grows more rapidly than other LAB over a range of temperatures (5-35C) and brine concentrations (0-5%)
• L. mesenteroides carries out heterolactic fermentation of vegetable sugars, typically sucrose, fructose, and glucose and produces carbon dioxide and acids (lactic and acetic)
• The rapidly dropping pH inhibits the growth of spoilage microorganisms, L. mesenteroides will grow until an acidity level of 1.5-2.0% is achieved
• The carbon dioxide replaces air and provides the anaerobic conditions favorable for the stabilization of ascorbic acid and the natural color of the vegetables
• If the temperature is above 22C L. mesenteroides will not grow, and this will be detrimental to the development of the fermentation

Question 6

What are the successors of L.mesenteroides , at what conditions they grow, how the end of the log phase of this outcompeting organism is connected to cabbage quality

Answer 6

• The acidity produced by L. mesenteroides and other LAB eventually inhibits the growth of these heterofermentative microbes in favor of more acid-tolerant homofermentative LAB
• Lb. plantarum are able to survive the elevating lactic acid levels and produce exclusively lactic acid from the remaining sugars
• Above an acidity of 2.0% Lb. plantarum will grow
• In most vegetable fermentations Lb. plantarum will outcompete other LAB at the end of the reaction due to its superior acid tolerance
• At the end of the log phase for Lb. plantarum growth there is little sugar left, and vegetable ferment is at an acceptable quality to be served or canned

Question 7

Is there anyone that can continue fermenting after L.plantarum

Answer 7

•Lactobacillus brevis Will continue fermenting after L. plantarum until an acidity level of 2.5-3% is obtained, and there is no sugar left in the vegetables

Question 8

Is bacteriophage a problem in vegetable fermentation?

Answer 8

No

Question 9

Why bacteriophage is not a problem for vegetable fermentation?

Answer 9

• Dairy fermentations are carried out on pasteurized milk using a single starter culture of a cocktail of a few cultures → If a bacteriophage infects the starter culture, the fermentation will fail
• Vegetable fermentations, however, do not typically use starter cultures, but if phages are present and inhibit one strain of LAB, another (resistant) indigenous strain will grow instead

Question 10

How much glucose and fructose cabbage has

Answer 10

•Cabbage consistently contains 2.5% glucose and 2% fructose

Question 11

Give an overview of sauerkraut fermentation, do you need starter culture, for how long the fermentation will go, salt concentration needed, how long, what compounds are produced

Answer 11

• If given anaerobic conditions, at approximately 22C, and 2-3% salt concentration, LAB dominate the microbial community able to grow on the sugars from the cabbage
• High-quality sauerkraut can be produced without a starter culture if the conditions are correct
• During the first 24-48 hours of the fermentation, carbon dioxide gas, lactic acid, and to a lesser extent acetic acid will be produced from the salted cabbage

Question 12

What is the optimal temperature for sauerkraut production, why?

Cna you increase the temperature and speed up the process?

Three different categories of temperatures to produce sauerkraut

Answer 12

• Temperature is important, the best quality sauerkraut is produced between 18-22C, since this temperature favors the growth of L. mesenteroides
• Generally, lower temperatures produce higher quality sauerkraut
• Higher temperature can produce sauerkraut in 7-10 days but it will be of lesser quality, LAB don’t grow as well during fast fermentation producing a much less complex flavor•

Summary:

• Below 7.5C fermentation time can be up to 6 month, flavor good
• 18-22C fermentation time is ~20 days, optimal flavor
• 32-36C fermentation time is 10 days, flavor is poor

Question 13

Why the outer leaves are removed in salting the cabbage and why the core?

Answer 13

• The outer leaves are removed to lower the non-LAB bacterial load
• The core contains high levels of sucrose, which can lead to dextran formation by L. mesenteroides resulting in a slimy texture (Yuck!), so it is also removed

sucrose is turned into dextrin-> slimy, bad texture

Question 14

Why salt is added to sauerkraut (2 objectives)

Answer 14

• To extract cabbage juice from the leaves to make the sugars and nutrients from the plant material readily available to the LAB
• To keep non-LAB from spoiling the cabbage. LAB are resistant to moderate amounts of salts, non-LAB are less resistant

Question 15

How the oxygen is removed for sauerkraut and why

Answer 15

• Remove oxygen, have an air lock, submerge cabbage in brine
• Removing the oxygen favors the growth of LAB, over the growth of molds

Question 16

How the proportion of pathogens and good bacteria is changing within a day of the start of sauerkraut fermentation? How it is translated into MRS and TNTC plates

Answer 16

• During the first few days in the ferment the levels of Enterobacteriaceae drop 5-log
• Enterobacteriaceae are known enteric pathogens
• While the aerobes and the LAB increase
• Several groups observed this first hand in the lab
• 0 hr the MRS plates were TNTC (aerobes)
• 24 hr there were a few colonies on MRS plates

Question 17

How the sugars levels is altered during the first 24 hours of sauerkraut fermentation

Answer 17

• Sucrose remains at low levels
• Fructose is rapidly used
• Glucose increases for the first few days
• Water is released from the leaves faster than glucose
• The increase in glucose is not microbiological but instead is the slow release of the glucose from the cabbage leaves

Question 18

How the acidity and pH is changed in sauerkraut fermentation?

Answer 18

• The total titratable acidity increases rapidly, slows, then increases again, but at a slower rate
• The increases in titratable acidity corresponds to a decrease in pH
• This isn’t the case for all fermentations

Question 19

why mannitol increases during sauerkraut fermentation

Answer 19

• Heterofermentative lactic acid bacteria use fructose as an electron acceptor converting it to mannitol
• Mannitol remains constant during extended fermentation

Question 20

What two acids are produced by heterolactic acid bacteria and their contribution

Answer 20

• The initial heterolactic stage of the fermentation results in the production of both lactic and acetic acids
• The acetic acid is volatile and makes an important contribution to the flavor of the final product

Question 21

What final concentrations of mannitol, acetic and lactic acids in the end

Why the fermentation stops?

Answer 21

• The fermentation end point (21 days) includes mannitol and acetic acid at ~1% each and lactic acid at ~2%.
• There is still sugar in the final sauerkraut, acid accumulation is the limiting factor

Question 22

Why there is a shoulder at around day 8?

Answer 22

•The shoulder at ~8 days is when the heterofermentative LAB die, and the second growth curve (diphasic growth) is created when the homofermentative bacteria become dominant

Question 23

Why sauerkraut color on the top is different that on the bottom?

Answer 23

• Ascorbic acid concentration decreases a little during the first days of the fermentation if the oxygen in the head space is not pumped out
• This is because it is oxidized by dissolved oxygen from the headspace
• If ascorbic acid remains high the sauerkraut retains a light straw colour, if the ascorbic acid decreases the kraut has a grey/brown colour.
• If your compare the colour of the sauerkraut on the bottom of your mason jar with the sauerkraut on the top you should see somewhat of a colour differential
• This does not effect taste

Question 24

For how long commercial sauerkraut can be stored in US/canadam what is the risk?

Answer 24

• In Canada/ US commercial sauerkraut can be stored in bulk tanks for up to 1-year until it is processed for food service or retail sale
• A danger is that the product may sour as lactic acid continues to accumulate

Question 25

When commercial sauerkraut is packaged in Europe? What taste do they like?

Answer 25

•In Europe sauerkraut is packaged after about 1 week and is preferred to have a mildly acidic taste

Question 26

Is something done to commercial sauerkraut after production and before canning?

Answer 26

* **Most commercial sauerkraut products are pasteurized and then canned, killing all of the LAB (any heating above 41C will kill most LAB)** * It also ”cooks” the cabbage making it much less crispy•If you purchase refrigerated sauerkraut there is a possibility that some LAB will remain

Question 27

The primary ingredients for kimchi?

Answer 27

* Chinese cabbage, and Asian radishes are used as the primary ingredients * Small amounts of other ingredients: garlic, ginger, anchovies, ang other vegetable material, can be added

Question 28

How the kimchi process is different from sauerkraut? Mention salt % of brine, the temperature of fermentation, final acid content

Answer 28

* For cabbage kimchi (baechu) the fresh cabbage is cut in half and soaked in 5-10% brine to wilt the cabbage, the cabbage is drained, and a paste of red pepper is added * The ingredients are packed into jars and held in “kimchi refrigerators” which provide a temperature of 18C for a few days followed by cold refrigeration (1-2C) * This allows the heterolactic fermentation to take place, but halts the reaction before the homofermentative stage occurs (keeping the kimchi from becoming as sour as sauerkraut) * Kimchi has 0.6% acid

Question 29

Types of kimchi

Answer 29

* White Kimchi (contains no red pepper) * Traditional Kimchi (which has the diphasic heterofermentative and homofermentative pattern of LAB succession)

Question 30

How vitamin levels are altered during kimchi and sauerkraut?

Answer 30

* Vitamin B content increases during sauerkraut and kimchi fermentation * Vitamins C and A are maintained

Question 31

Are all cucumbers fermented in the store?

Answer 31

•**Not all pickles are fermented pickles, some can be pasteurized and canned in acid**

Question 32

How cucumbers are fermented in industrial scale in US and canada

Answer 32

* In the US and Canada fermentations are done in 30,000 liter, open-top, fiberglass tanks located outside so the surface is exposed to sunlight * The UV radiation from the sunlight kills aerobic surface yeasts * Cucumbers are covered with salt brine and held below the surface with wooden headboards

Question 33

What is final NaCl of cucmbers and Calcium chloride and why

Answer 33

•Fermentations generally have 6% NaCl and 0.1-0.4% calcium chloride to maintain the crisp texture of the fermented cucumber

Question 34

What is the usual type of bacteria that does cucumber fermentation, why?

Answer 34

* Cucumber fermentations typically undergo homolactic fermentation, which does not result in the production of carbon dioxide * The high salt concentrations favor homofermentative LAB

Question 35

Why air pockets can be created in cucumber fermentation? what can be done to prevent it

Answer 35

* Carbon dioxide may be generated from the respiration of cucumbers and by the **decarboxylation of malate** during the beginning of the fermentation reaction * Some LAB have inducible **malolactic enzymes** that converts **malate to lactate and carbon dioxide** * While this is desirable in the production of some wines, it may result in “bloaters” during cucumber fermentation * This happens when bacteria produce internal gas pockets in fermented cucumbers•In an effort to prevent bloater formation, cucumber fermentations are purged with air to remove excess CO₂ from the tank * **Potassium sorbate or acetic acid** can be used to limit the growth of aerobic organisms in air purged fermentations

Question 36

Is cucumber fermentations spontaneous? Can be it with the starter culture, wh? any other methods of starting fermentation

Answer 36

* Most commercial cucumber fermentations rely on the growth of LAB naturally present on the cucumber, although the trend is now starting to be to use a *Lb. plantarum* starter * A commercial *Lb. plantarum* that does not decarboxylate malic acid (no bloaters) has been developed * Several commercial fermentations use recycled brine (cheaper and serves as a starter)

Question 37

For how long fermented cucumbers ca be stored, any changes in NaCl content due to geographical location? ARE cucumbers pasteurized

Answer 37

* After the fermentation, the cucumbers can be stored in fermentation tanks for 1-year * In geographic areas where the temperature drops below zero, the concentration of NaCl is often increased to minimize freezing damage. * Some fermented cucumbers are pasteurized prior to sale, but further microbial growth is generally prevented by organic acids, low pH, and lack of fermentable sugars

Question 38

What is the final lactic acid content and pH, and sugar of fermented cucumbers

Answer 38

* At the end of the fermentation there may be 1.5% lactic acid, a pH of 3.0, and no residual sugar * Only a few types of microorganisms can grow in this environment

Question 39

Can fermented cucumbers be spoiled? Why

Answer 39

* Occasionally, fermented cucumbers can undergo a secondary fermentation that spoils the product * In this secondary fermentation the pH increases, the lactic acid decreases, and propionic and butyric acids are formed – which causes the product to stink * The microbial ecology of this type of spoilage is not defined, but when the pH increases above 4.6, in theory, *Clostridium botulinum* could be an issue

Question 40

Three types of commercial olives (mention fermented or not)

Answer 40

* There are three principle types of commercial olives * Green table olives (fermented) * Natural black olives (fermented) * Canned ripe black olives (not fermented)

Question 41

WHAT IS DONE TO table green olives prior to fermentation, why?

Answer 41

•Green table olives are treated with lye (1-3% NaOH) prior to fermentation•The strong base treatment serves multiple purposes: ## Footnote * It reduces the natural bitterness of olives * It decreases the natural antimicrobial activities of the phenolic components of the olives * It makes the skin of the olive more permeable allowing the sugar to diffuse out during fermentation

Question 42

What happens after alkali treatment for green table olives? What are microorganisms doing fermentation and why

Answer 42

* Olives are washed and brined (10% NaCl) after alkali treatment * Due to the strong base treatment the initial pH of the fermentation can be above 7, and this means that the first microflora are generally resistant to alkali conditions (*Bacillus sp. Enterobacter sp. Citrobacter sp.*) * As the pH drops below 6, LAB colonize then dominate the fermentation specifically *Lb. plantarum*

Question 43

What is the difference in fermentation between green and black olives?

Answer 43

* Natural black olives are also prepared by fermentation, but they do not receive NaOH treatment prior to brining * They are picked in a ripened state and have a softer texture than green table olives * Fermentation is a much slower process with black olives because of the lack of NaOH treatment: * Antimicrobial phenolic compounds dissolve into the brine, slowing fermentation * Diffusion of sugars out of the olives is slower in black olives * As a result this fermentation can take months to complete