Microbiology of ice cream

 

Microbiology of ice cream

Ices were known in ancient times and Chinese mixed snow and fruit juices to make an iced sweet and ice cellars were used to keep foods cool.  Currently, different types of frozen dessert are available such as Cream Ices, Ice Cream, Milk Ices, Custards, Ices or Water Ices, Sherbet, Sorbets, Mousse, Cassata, Splits, Frozen Yogurt, etc.

Ice cream and other whipped frozen desserts are foams made up of air cells surrounded by a partially frozen emulsion. Ice crystals and solidified fat globules are embedded in the continuous unfrozen liquid phase that contains proteins, carbohydrates, salts, and gums. Ice cream has water, fat, emulsifiers, milk solids non-fat (MSNF), sugars, stabilizers, air, and ice as its major components.

Manufacture of ice cream

The manufacture of ice cream is a complex operation with a number of steps.

1. The ingredients will be weighted or measured into the mixing vessels.

2. After mixing, the ingredients are pasteurized, either heat-treated in a batch system or pumped to HTST continuous-flow pasteurization

3. it will be then homogenized in a homogenizer and homogenization will reduce the size of the fat globules, producing a uniform and stable suspension of fat in the mix. The organoleptic characteristics of the product are improved by providing the correct body and texture, mouth feel, and appearance.

4. After pasteurization and homogenization, the mix is rapidly cooled to 2-4°C in order to preserve the bacteriological quality of the mix. The mixture is then stored for approximately 24h, and during

this period of “aging”, the milk proteins hydrate, the fats begin to crystallize, and any added hydrocolloids absorb quantities of water.

5. The mix is then passed to the ice cream freezer where its temperature is reduced rapidly, and at the same time the mix is subjected to considerable agitation.

6. The ice cream is packaged and then kept at a temperature of about -30°C until, and during, distribution.

Microbiology of Icecream

In the freezer, the temperature of the mix is rapidly reduced, air is incorporated and vigorous whipping of the ice cream is done. Both intra- and extracellular ice crystals mechanically injure frozen cells. Ice crystals that form outside the cell reduces the amount of free water and those that form inside the cells puncture cell membranes.

Once the ice cream leaves the freezer, it is then held at temperatures around -20°C or lower until it is consumed.  Pathogenic organisms have been shown to survive for years in ice-cream.  Therefore, it is essential that the bacteriological content of the ice cream from the freezer should be as low as possible and pathogenic organisms should be absent.

Many factors affect the microbiological quality of the finished product, including the relatively complex manufacturing process and the equipment that are used for the processing. Liquid milk, cream, and skim-milk concentrate should be subjected to adequate heat treatment.  Granulated sugar should be almost sterile. Butter and anhydrous milk fat should preferably be stored at a temperature less than  -20°C and Vegetable fats and Stabilizers and emulsifiers should not present any problems. Many other foodstuffs added to ice cream, such as fruits, nuts, chocolate, broken biscuit, colors, and flavors should be of satisfactory microbiological standard and only pasteurized eggs must be used if they are added after the heat treatment of the ice cream mix. Colors and flavors must also be handled with great care to avoid contamination. Air that incorporated into the ice cream must be filtered so as to ensure that no contamination occur. The packaging material should also be free from contamination.

The heat treatment applied to the ice cream is more severe than the pasteurization of the milk and thus is more efficient in killing microbes.  Any contamination that may occur during the handling of ingredients, packing materials, and the product during processing and distribution must be eliminated, or kept to the very minimum. The equipment that is most difficult to clean and disinfect includes the freezers and the homogenizers and mix holding tanks and the tanks used for the storage of raw materials.

 

References

Dairy Microbiology Handbook, Third Edition, Richard K. Robinson, Wiley Interscience publications

Probiotics and Prebiotics

Probiotics and Prebiotics

Probiotic means “for life” (Latin and Greek) and probiotic bacteria influence general health and well-being through their association with the gastrointestinal tract (GIT) and its normal microbiota. The concept of probiotics was first coined by Elie Metchnikoff.  He proposed that a normal, healthy gastrointestinal microbiota in humans and animals provided resistance against “putrefactive” intestinal pathogens and he isolated a Lactobacillus culture from a fermented milk which provided health benefits.

Our body are colonized by thousands of microbial species that exist as commensals, largely on mucosal tissues of the nose, mouth, GIT, and vagina.  Some of the most widely used probiotic species are part of the natural commensal microbiota. They have proteins helping them for metabolic activities for sugar harvesting and adherence to host.

10¹⁴ bacterial cells are found in the human body, 10-fold more cells than the 10¹³ mammalian cells comprising the human body itself.  Most of these bacterial cells reside in the GIT.  The composition of the GIT microbiota is complex, dynamic, and specific to each host and change markedly with diet, age, and lifestyle. They are responsible for many important properties that affect the metabolism of food and drugs, the renewal of gut epithelial cells, immune system development, heart size, and general behavioral characteristics. The impact of the microbiota on the host can depend on many factors such as diet, age, exposure to exogenous microorganisms, the genetic makeup of the host, and physiological conditions of the host tissues.

The digestive tract is composed of four major categories

·         Autochthonous microbiota: populations of microbes that have permanently colonized the host and are present in large numbers

·         Normal microbiota: microorganisms that are frequently present but can vary in number and be sporadically absent

·         Pathogens: microorganisms that are periodically acquired but can persist and cause infection and disease

·         Allochthonous microbiota: microbes of another origin that are present temporarily (most probiotic cultures are allochthonous)

Only a small proportion of the GIT microflora is culturable. Molecular biology techniques are used for their studies. Each person has a persistent flora that can be recovered repeatedly over extended periods. Severe disturbances to the GIT, such as antibiotic therapy, enteric infections, or dietary stress, can temporarily disrupt the autochthonous and normal microbiota. However, these populations quickly recover.

Lactobacillus and Bifidobacterium species are natural residents of the GIT and are beneficial to the health of their hosts and pose no risk to the host. Some species of Bacillus and Escherichia are also proposed as probiotics, but the two major species around which the probiotic concept has evolved are Lactobacillus and Bifidobacterium.

Probiotics is a substance produced by one microorganism that stimulates the growth of another microorganism. It is a live microbial supplement which improves the host intestinal balance and is beneficial to the host. There are many types of probiotic cultures (mono- and mixed strain cultures, multiple probiotic species), applications (gastrointestinal versus topical), and mechanisms of probiotic activity (live cells, dead cells, and cellular components). According to WHO they are “live microorganisms which when administered in adequate amounts confer a health benefit on the host”.

Abiotics are probiotic strains that have been inactivated by lysis, heat, or UV irradiation and have some effectiveness in downregulating inflammatory response in intestinal epithelial cells.

The mechanisms through which probiotics exert their beneficial effects are

1.      Interaction with the immune system. Some probiotics can alter cytokine production by exposed macrophages and dendritic cells and can shift the production of cytokines from the inflammation-inducing interleukin- 12 (IL-12) pathway to the anti-inflammatory IL-10 pathway

2.      Strengthening the mucosal barrier. The GIT functions as a semipermeable barrier that allows the selective passage of certain molecules. Dysfunctional barriers are involved with diseases of the GIT such as inflammatory bowel disease. Probiotic strains reinforce and repair this barrier by stimulating the production of protective proteins, such as mucins, by intestinal epithelial cells.

3.      Exclusion of pathogens. Probiotic strains can inhibit the adherence of bacterial and viral pathogens and prevent infection by them.

Probiotics are used for Animal Nutrition, for Human Health and as Therapeutics for treatment of diseases, including the treatment of diarrheal conditions, inflammatory bowel disease, allergies, and infections of the upper respiratory tract.

 

Prebiotics

Prebiotics are ingredients that selectively promote the growth of the existing beneficial microbiota. Growth factors in human breast milk, which stimulate the growth of Bifidobacterium is an example.

Prebiotic is a nondigestible food ingredient that beneficially affects the host by selectively stimulating the growth and/or activity of one or a limited number of bacteria in the colon and Synbiotic is a combination of a prebiotic ingredient with a probiotic culture.

Food ingredients classified as prebiotics exhibit the following characteristics:

·         Limited hydrolysis and absorption in the upper GIT

·         Selective growth stimulation of beneficial bacteria in the colon

·         Potential to repress pathogens and limit virulence (by attenuation of virulence, immunostimulation, and stimulation of a beneficial flora)

The best-known prebiotics are fructo-oligosaccharides derived from food sources. The largest natural source is inulin from the chicory root which is also found in edible plants like onions, asparagus, bananas, wheat, etc.

Examples of other prebiotic factors are Oligosaccharides, Fructans, Human milk, Lactitol, Synthetic derivative of lactose, etc

 

References

Food Microbiology: Fundamentals and Frontiers, 4^th  Edition, Michael P. Doyle, Robert L. Buchanan, ASM Press, Washington, DC 20036

Single Cell Protein

 Single Cell Protein

Single Cell Protein (SCP) are protein derived from microorganisms.  The biomass or protein extract from pure or mixed cultures of algae, yeasts, fungi or bacteria is used as an ingredient or a substitute for protein-rich foods.  This is suitable for human consumption and as animal feeds.

It emerged in the 1950s and 1960s as an alternate and unconventional source of food to bridge the ‘food gap’ between the industrialized and the less industrialized parts of the world, especially as a protein source.

SCP has a number of attractive features:

·         It is not subject to weather variations and can be produced throughout year

·         Microorganisms have a much more rapid growth than plants or animals.

·         Waste products can be turned into SCP.

Disadvantages of SCP

·         Lack of expertise and/or the financial resources to develop fermentation industries in developing countries, where protein malnutrition exists

·         Microorganisms contain high levels of RNA, consumption of which lead to uric acid accumulation, kidney stone formation and gout.

During the First World War, Saccharomyces cerevisiae, were grown on a molasses-ammonium medium. Geotrichum lactis, Endomyces vernalis, and Candida utilis were grown for food.

A wide variety of substrates are used for SCP production such as hydrocarbons, alcohols, and wastes from various sources.

Hydrocarbons

a. Aliphatic hydrocarbons are assimilated by strains of yeasts while other classes of hydrocarbons, including aromatics are not efficiently assimilated.

b. n-Alkanes of chain length shorter than n-nonane are not assimilated and Yield factors increase with increasing chain length.

c. Unsaturated compounds are degraded less readily than saturated ones and branched chain compounds are degraded less readily than straight chain compounds.

Among the gaseous hydrocarbons, methane has been most widely studied as a source of SCP and propane and butane are also studied.   Single cell protein production from methane use continuous cultures and a mixed population of microorganisms and the advantages are higher growth rate, higher yield coefficient, greater resistance to contaminations and a reduction in foaming.

The four-organism mixture is a fast growing mixture - the unnamed methane bacterium utilizes methane and produces methanol, Hyphomicrobium utilizes the methanol and Flavobacterium and Acinetobacter remove waste products.

The major source of liquid hydrocarbons is crude petroleum which is highly variable in composition. The petroleum hydrocarbons which are used to grow SCP are diesel oil, gas oil, fuel oil, n-alkanes (C10 - C30 and C14 – C18, C11 – C18, C10 - C18) n-hexadecane n-dodecane.

Due to the crude oil price rise, the use of crude oil as a substrate for SCP is on a decline.

Methanol is suitable as a substrate for SCP for the following reasons:

(a) it is highly soluble in water

(b) the explosion hazard of methanol is less

(c) it is readily available in a wide range of hydrocarbon sources

(d) it can be readily purified

(e) it requires less oxygen than methane for metabolism by micro-organisms

(f) it is not utilized by many organisms.

Several companies in Italy, West Germany, Norway, Sweden, Israel, the United Kingdom, and the United use methanol as a SCP substrate. Example is Imperial Chemical Industries (ICI) in UK which use the bacterium, Methylophilus methylotropha to produce ‘Pruteen’ using the loop fermentor.

Hansenula, Pichia, Torulopsis and Candida grow on methanol.

Ethanol can be utilized by many bacteria and yeasts and as a substrate for SCP, it is used by yeasts. Ethanol has the following advantages:

(a) It is like methanol, highly miscible with water

(b) Can be more safely stored and transported

(c) Unlike methanol, it is non-toxic it can be more easily handled

(d) Ethanol is partially oxidized, requires less oxygen

The major disadvantage in using ethanol for SCP production is that it is expensive

Candida utilis. Hansenula anomala, Acinetobacter caloaceticum grow using ethanol

Waste Products

Due to the hike in petroleum prices, substrates derived from plants which are renewable are used as substrate for SCP.

(i) Plant/wood wastes: corn cobs, plant stems, leaves, stalks, husks, etc  are cellulose containing materials. Pretreatment such as ball-milling, acid, alkali, sodium chlorate or liquid ammonia treatment is needed to make cellulose susceptible to fermentation and lignin must be broken down. 

(ii) Starch-wastes: Starch-containing wastes from rice, potatoes, or cassava industry can be used for SCP production. Starch hydrolysis is relatively easy.  In Symba Process developed by the Swedish Sugar Corporation, two yeasts are used symbiotically - Endomycopsis fibuligera hydrolyses starch to glucose and maltose and Candida utilis utilizes these sugars.

(iii) Dairy wastes: Whey, by-product of diary industry is liquid rich in lactose.  Saccharomyces fragilis is grown in it to produce either SCP or alcohol.

(iv) Wastes from chemical industries: C. lipolytica or Trichosporon cutaneum can be used for SCP production in oxanone water, a waste mixture of organic acids from the copralactam used for the manufacture of nylon.

(v) Miscellaneous substrates: Molasses, the by-product of the sugar industry is used for production of SCP.  Coffee wastes, coconut wastes, palm-oil wastes, citrus waste, etc can also be used.

Microorganisms used in SCP production

Organisms to be used in SCP production should have the following properties:

(a) Absence of pathogenicity and toxicity

(b) Protein quality and content should be high

(c) Digestibility and organoleptic qualities

(d) Must grow rapidly in a cheap, easily available medium.

(e) Adaptability to unusual environmental conditions such as low pH conditions or high temperature

The heterotrophic microorganisms currently used are bacteria and fungi.  Protozoa are not used in SCP production. The gaseous hydrocarbons are used by bacteria and liquid hydrocarbons and alcohols are utilized by both bacteria and yeasts. Cellulose in peanut shells, carob beans, spoiled fruits, corn and pea wastes, sugarcane bagasse, palm, cassava wastes are used to make SCP using Trichoderma sp., Glicladium sp., Geotrichum sp., Fusarium, and Aspergilus.  Fungi are lower in RNA content and are easily harvested.

Autotrophic organisms such as photosynthetic bacteria and algae are used as SCP. The disadvantage with photosynthetic bacteria is that they require anaerobic conditions for photosynthesis which is difficult to provide and maintain.

Algae have high protein concentration, greater than soya bean and dietary energy from algae is higher than that of sugar beet, corn and potato.  For high algal yields carbon dioxide is supplied to algae growing in day light.  Where saline water rich in bicarbonates is available, supplementation with CO2 is not necessary.  Effluents from sewage treatment are ideal for growing algae for animal feed, where the algae should be heat-treated to avoid any possibility of pathogen transmission.

Algal cultivation is easier and it is highly digestible by ruminants and other animals.

Microbes employed include:

Yeast - Saccharomyces cerevisiae, Pichia pastoris, Candida utilis, Torulopsis coralline, Geotrichum candidum

Fungi - Aspergillus oryzae, Fusarium venenatum, Sclerotium rolfsii, Polyporus, Trichoderma

Bacteria - Methanomones sp., Methylococius capsulatus, Pseudononas sp., Flavobacterium sp. Arthrobacter simplex, Nocardia paraffinica, Nocardia paraffinica, Rhodobacter capsulatus, Rhodopseudomonas glatinosa

Algae – Spirulina, Chlorella

Concerns of using SCP

Due to the novelty of SCP as food receives strong opposition especially in Japan and Italy where the government is concerned with the possibility of the presence of carcinogenic compounds in petroleum-grown SCP, content of nucleic acid in SCP, the polycyclic aromatic hydrocarbons and the presence of n-paraffins, etc

Protein Advisory Group (PAG) formed by WHO in 1955 concluded that low levels of residual alkanes, the presence of odd-number fatty acids, or polycyclic hydrocarbons derived from petroleum do not possess a danger in terms of carcinogenicity or toxicity. They also developed guidelines for the production and nutritional and safety standards of SCP for human consumption.  These include microbiological examination for pathogens and toxin producers, chemical analyses for heavy metals, nucleic acid content, presence of hydrocarbons, safety tests on animals and protein quality studies.

Another problem associated with SCP is the nucleic acid content.  When nucleic acid is eaten by man, it is broken up by nucleases present in the pancreatic juice, and converted into nucleosides by intestinal juices. Guanine and adenine are converted to uric acid. As a result, when foods rich in nucleic acid are consumed in large amounts, uric acid level increases in blood plasma resulting in its deposition in various tissues in the body including the kidneys and the joints and kidney stones and gout may result.

Various methods for the removal of nucleic acids from SCP are

(a) Growth and cell physiology method: The RNA content of cell is dependent on growth rate.  The growth rate is reduced to reduce nucleic acid.

(b) Extraction with chemicals: Dilute bases such as NaOH or KOH will hydrolyze RNA easily. Hot 10% sodium chloride may also be used to extract RNA and the protein may then be extracted, purified and concentrated.

(c) Use of pancreatic juice: RNAase from bovine pancreatic juice, which is heat-stable, can be used to hydrolyze yeast RNA at 80°C.   At this temperature the cells are more permeable.

(d) Activation of endogenous RNAase: The RNAase of the organism is activated by heat-shock or by chemicals to reduce the RNA content of yeasts.

The nutritional value of SCP depends on the composition of the microbial cells used, especially their protein, amino acid, vitamin, and mineral contents. SCP derived from bacteria and yeasts is deficient in methionine. Glycine and methionine are sometimes deficient in molds. These can be improved by supplementation with small amounts of animal proteins.

 

References

1. Modern Industrial Microbiology and Biotechnology, Nduka Okafor, Science Publishers

 

Fermented Vegetables

 Fermented Vegetables

Vegetables were preserved by fermentation from time immemorial. A wide range of vegetables and fruits including cabbages, olives, cucumber, onions, peppers, green tomatoes, carrots, okra, celery, and cauliflower are preserved. The fermentation process for vegetables result in nutritious foods that may be stored for extended periods without refrigeration. Before fermentation, fresh fruits and vegetables contain a variety of microorganisms, including aerobic spoilage bacteria, yeasts and molds. Their growth deteriorates the vegetable material, due to the degradative enzymes such as proteases, lipases, amylases, nucleases, etc released from these microbes.  Brining vegetables for fermentation results in the production of organic acids, CO₂ and a variety of antimicrobial compounds by lactic acid bacteria.  The production of acid quickly lowers the pH which inhibit the development of undesirable microorganisms and their enzyme activity, carbon dioxide produced provides anaerobic conditions which favors the stabilization of ascorbic acid and the natural color of the vegetables.

Vegetables are fermented through either of the following methods

·         Dry salted – vegetable is treated with dry salt and the juice from the vegetable forms the brine.  Generally, for 100 kg vegetable 3 kg of salt is needed. 

·         Brine salted – Brine solution of 15-20% salt is used in which the vegetables are kept. 

·         Non-salted – vegetables are fermented by lactic acid bacteria without addition of salt

Biochemistry of vegetable fermentations: During the fermentation of cucumbers, cabbage and olives, glucose and fructose are converted to lactic acid, acetic acid, ethanol, and CO₂ by LAB and yeasts. The primary pathway for homofermentative LAB involves the breakdown of six-carbon sugar (glucose) to give three-carbon lactic acid molecules. Heterofermentative organisms use a more complex metabolism. Glucose is initially converted to CO₂ and a five-carbon sugar phosphate, which is further degraded to lactic acid and a two-carbon compound, ethanol or acetic acid.

Cabbage Fermentations – Sauerkraut and Kimchi

Examples of commercial production of fermented cabbage are kimchi in Korea (made from the Chinese cabbage, Brassica rapa) and sauerkraut in the United States and Europe (made from Brassica oleracea).

Sauerkraut

Cabbage is sliced into thin pieces known as slaw and preserved in salt water or brine containing

about 2.5% salt at a temperature of about 15°C. The slaw must be completely immersed in brine to prevent it from darkening. Fermentation is initiated by Leuconostoc mesenteroides which is a heterofermentative lactic acid bacterium which can grow over a wide range of pH and temperature.  The CO₂ generated during fermentation will create an anaerobic condition that eliminate other microorganisms and encourages the growth of other lactic acid bacteria such as L. brevis, L. plantarum, Pediococcus cerevisiae, etc. Coliforms and pseudomonads disappear.  Lactic acid bacteria produce compounds such as lactic and acetic acids, ethanol, and volatile compounds such as diacetyl, acetaldehyde, acetal, isoamyl alcohol, n-hexanol, ethyl lactate, ethyl butarate, and iso amyl acetate which contribute to the flavor of sauerkraut

Sauerkraut fermentations are done in large fermentation tanks that contain 100 tons or more of shredded or chopped cabbage. Cabbage contains 4 to 5% sugar (2.5% glucose and 2% fructose).  The shredded cabbage is dry salted and conveyed to fermentation tanks, brine with an NaCl concentration of about 2 to 3% will be formed in the fermentation tanks. During the first 24 to 48 h, carbon dioxide and lactic and acetic acids are produced through the initial heterolactic fermentation. The volatile acetic acid contributes to the flavor and aroma. Heterofermentative microorganisms die off after one week and are replaced by the acid-tolerant homofermentative microorganisms. Sauerkraut may be stored for up to 1 year in fermentation tanks until it is processed for food service or sale.

It is reported that two heterolactic species, L. mesenteroides and Lactobacillus brevis, and two homolactic species, Pediococcus cerevisiae and Lb. plantarum are the bacteria present in the fermentation.

The salt serves a number of purposes in fermented cabbage

(i) it extracts moisture from the shredded cabbage by osmosis to form the brine in which the fermentation take place

(ii) it inhibits some of the natural microflora of the cabbage which may otherwise cause spoilage and helps to selective growth of the lactic acid bacteria

(iii) it helps to maintain the crisp texture of the cabbage by withdrawing water and inhibiting endogenous pectinolytic enzymes

(iv) It contributes to the flavour of the product.

Kimchi

Kimchi fermentation is microbiologically similar to sauerkraut fermentation, except the ingredients, flavor, and preparation methods. For cabbage kimchi, the fresh cabbage is cut in half lengthwise or quartered and initially soaked in brine of 5 to 10% NaCl to wilt the cabbage. The cabbage is then washed and drained. An aqueous paste of ground red pepper is prepared and mixed in with the cabbage leaves. Small amounts of additional ingredients, such as garlic, ginger, and jeotgal (a highly salted (20% NaCl) anchovy (a small fish)) and green onion are usually included.  Kimchi fermentation also has a biphasic heterofermentative and then homofermentative pattern of LAB succession, with a few prominent species (L. mesenteroides, P. cerevisiae, Lb. brevis, and Lb. plantarum).  After the fermentation, the final salt concentration is between 3 and 6%.

In rural areas, kimchi is traditionally packed into earthen jars and buried in the soil and in urban South Korea, kimchi is prepared using household kimchi refrigerators, which are small, programmable refrigerators that provide an initial 18°C fermentation period of a few days, followed by very cold refrigeration (1 to 2°C).

More than 65 different types of kimchi are identified based on differences in raw materials and processing.

The vitamin B content increases during sauerkraut and kimchi fermentations, and vitamin C and A are preserved.

Olive Fermentations

There are several methods for fermenting olives such as green table olives, natural black olives in brine, and canned ripe black olives.

Green table olives are treated with lye (1 to 3% NaOH) and then washed prior to being brined and fermented. The NaOH treatment reduce the natural bitterness of the fruit, due to the degradation of oleuropein, reduces the antimicrobial activity of the phenolic components of olives and makes the skin of the olive more permeable allowing sugar diffusion to improve fermentation. After NaOH treatment, the olives are washed and brined in 10% NaCl for fermentation. The initial microflora during fermentation include a variety of gram-positive bacilli (Bacillus species) and gram-negative enteric bacteria (Enterobacter, Citrobacter, Klebsiella, and Escherichia).  As organic acids accumulate and the pH decreases below 6, the LAB such as Lb. plantarum and Leuconostoc mesenteroides dominate the fermentation and yeast species such as Candida, Pichia, Saccharomyces, and others are also present. They contribute desirable flavor characteristics. Following fermentation, they are pitted and stuffed before sale.

Natural black olives are prepared by a slow fermentation without lye. For this black olives are picked in a ripened state and have a black color as well as a softer texture than green table olives. Fermentation slower due to the lack of NaOH treatment. Antimicrobial phenolic compounds diffuse into the brine and diffusion of sugars is also reduced, which slows fermentation and it may take months to complete. The microflora is usually yeasts of the genera Saccharomyces, Hansenula, Candida, Torulopsis, Debaryomyces, Pichia, Kluyveromyces, and Cryptococcus.

Ripe black olives are prepared by darkening olives through oxidation in an alkali followed by washing and canning.  These are prepared from green or semiripened olives and are brined without an initial NaOH treatment. Following storage in brine for up to 1 year, the olives are subjected to oxidation treatments in the presence of 1 to 2% NaOH which blackens the olives.  They are then washed with water to remove NaOH and then canned in 1 to 3% NaCl brine. Sterilization is needed for black olives to prevent botulism, because the pH is generally above 4.6.

Sinki

This is a non-salted fermented radish tap root product traditionally consumed in some north-eastern states of India, Nepal and Bhutan. Fermentation is carried out by Lactobacillus plantarum or Lactobacillus brevis.  It is the most popular pickle in Nepal.

Fresh radish roots are washed and wilted in sun light for 1-2 days.  They are shredded, washed and packed in a jar and kept for fermentation at 30^oC for about 12 days.  During this period, L fermentum and L brevis followed by L plantarum carry out fermentation and drop the pH to 3.3.  After fermentation, it is sundried to remove moisture content and processes in a clay line pit for 2-3 months.

Gundruk

This is a non-salted fermented vegetable made from the leafy vegetables such as Mustard, Cauliflower and raddish. This is used in Nepal.  Shredded leaves are packed in an earthen pot with wamwater covering the leaves in a warm place for 5-7 days.  After fermentation it is sundried.  Pediococcus pentosaceus, Lacobacillus cellobiosus and Lacobacillus plantarum  are the organisms involved. 

Sunki

This is a non-salted fermented vegetable prepared from the leaves of Otaki Turnip in Japan.  The otaki-turnip is boiled and mixed with a wild small apples and dried sunki from previous year and kept for fermentation for 1-2 months at a low temperature, preferably during winter season.  The microorganisms involved in fermentation are Lactobacillus plantarum, Lactobacillus brevis, Bacillus coagulans and Pediococcus petosaceus

Kawal

This is fermented leaf preparation from a Wild African Legume Cassia obtusifolia.  This is highly rich in protein and used a meat analogue.  The leaves are not washed, ground into a paste and kept in earthen ware pot for 14 days.  The contents are mixed once in three days.  Microorganisms involved are Bacillus subtilis, Propionibacterium, Lactobacillus plantarum, Candida krusei, Sacharomyces, Rhizopus, etc.

 

Cucumber Fermentations (Pickling)

The word “pickle” usually refers to a pickled cucumber.

Cucumber pickles may be prepared unfermented, partially fermented or fully fermented.  They are then heated so that the interior of the cucumber reach 73.9^OC for 15 minutes.  Cucumbers (Cucumis sativus) for pickling are harvested before they are mature. Mature cucumbers are generally not fermented since they are large, ripen easily and are full of mature seeds.  Cucumber fermentations typically undergo a homolactic acid fermentation by Lb. plantarum and related LAB, which does not result in production of carbon dioxide. 

Most commercial cucumber fermentations depend upon growth of the LAB that are naturally present on the surface of cucumbers. Sometimes starter cultures are used to enhance product consistency.  (Starter cultures consist of microorganisms that are inoculated directly into food materials to carry out fermentation to cause desired and predictable changes in the food product).

There are two types of fermented pickles – Salt or Salt-stock pickle and dill pickle. 

Salt or Salt-stock pickle

Immature cucumbers are washed, packed in barrels or tanks and brined.  Sometimes 1% glucose is added.  Depending upon the level of salt, two methods are there, high salt method and low salt method.  In the low salt method, a low amount of salt is added and the concentration is gradually increased until enough concentration is reached to prevent contaminating bacteria.  8% brine is added initially and 9 lb salt per 100 lb cucumber are added.  In high salt method, 10.5% brine is added initially and 9 lb salt per 100 lb cucumber are added. 

During the primary fermentation for about two or three days, most of the unwanted bacteria disappear and the lactics and yeasts proliferate. Initially there will be Pseudomonas, Flavobacterium, Bacillus spp, Leuconostoc mesenteroides, streptococcus fecalis, etc.  In the final stages, after 10 to 14 days, Lactobacillus plantarum and L. brevis, followed by Pediococcus, are the major organisms.  The traditional process takes 6-9 week for completion. 

Commercial cucumber fermentations are commonly done in 30,000- to 40,000-liter, open-top, fiberglass tanks exposed to sunlight.  The UV radiation in sunlight kill aerobic surface yeasts that can metabolize lactic acid produced by the fermentation. Cucumbers are covered with salt brine (6% NaCl) and held below the brine surface. Calcium chloride (0.1 to 0.4%) is added to maintain the firm, crisp texture of the fermented cucumbers during fermentation and storage. After fermentation cucumbers may be stored in the fermentation tanks for 1 year or more. Fermented cucumbers are washed to remove excess salt and then packed with an appropriate cover liquor which contains acetic acid and spices in addition to residual lactic acid. Further microbial growth is prevented by the organic acids, low pH, and lack of fermentable sugars.

Dill pickles

These are named so because they are flavored by the addition of a herb, dill and some other spices, garlic and onion may also be added.  Here a lower concentration of salt is used and the brine is acidified using vinegar.  The low salt content allows more microbial production of acids. 

Overnight dill pickle

These are prepared by slow acid fermentation at low temperature in a low acidified brine.  The brined cucumbers are held at 3.3^OC when they undergo slow lactic acid fermentation and 0.3-0.6% acid is developed. These are having short keeping time. 

Genuine dill pickles

A brine containing 7.5-8.5% salt is added to cucumber and the concentration of salt in the finished pickle will be 3.5-4.5%.  Temperature between 15-30^OC is used.  The final acidity ranges from 1-1.5%.

Pickle defects and spoilage

·         Shriveling results from physical damage due to too much strong salt, vinegar or sugar solution.

·         Hollow pickles are formed when the cucumbers are left to stand for some time after harvesting and before fermenting.

·         Floaters or Bloaters - Some heterofermentative LAB form carbon dioxide during malolactic fermentation which result in the creation of bloaters.  These are fermented cucumbers with undesired internal gas pockets.

·         Slippery pickles – when cucumbers are exposed to air, capsulated bacteria may grow.

·         Soft pickles are the result of pectinolytic enzymes from molds or cucumber flowers.

·         Black pickles develop due to the formation of hydrogen sulfide by bacteria.

·         Ropy pickle brine is due to the growth of certain gram negative capsulated bacteria.

 

 

References

Food Microbiology: Fundamental and Frontiers, 4^th Edition, Michael P Doyle and Robert L Buchanan

Modern Industrial Microbiology and Biotechnology, Nduka Okafor, Science Publishers

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https://www.jstage.jst.go.jp/article/jgam1955/39/4/39_4_395/_article