STARTER CULTURES AND FERMENTED MILK PRODUCTS

Module 8. Forms of starters

Lesson 12

LIQUID, FROZEN, DRIED, CONCENTRATED AND DVS CULTURES

12.1 Forms of Starters

12.1.1 Liquid

Most popular and widely used form. The starters are normally preserved in small quantities. Working cultures are maintained with either weekly or daily sub-culturing. Starters can be preserved in liquid form using one of the following two different growth media viz.,Reconstituted skim milk, litmus milk. Reconstitute the Non-fat dry milk which is free from antibiotics to a total solid content of 10-12%. It is sterilized by auto claving at 10-15 psi / 10-15 minutes. Check the sterility by incubating at 30°C for one week.

Factors affecting the starter culture activity

1. The rate of cooling after incubation

2. Level of acidity at the end of incubation period.

3. The temperature of storage

4. Duration of storage

12.1.2 Litmus milk

Prepare the litmus milk containing the following composition.

It is sterilized by autoclaving at 10 psi/ 10 minutes. Check the sterility by incubating at 30°C for one week.Reactivate the cultures once in every 3 months

Disadvantages:

• Labour intensive

• Expensive

• Induces mutations

• Requires trained and skilled personnel

Lactococcus lact is ssp cremoris and leuconostocs can be sub-cultures up to 50 times without fear of mutation and they can be incubated at 22°C for 16-18 hours or at 30°C for 8-10 hours with1% inoculum. The yoghurt cultures can be sub-cultured up to 15-20 times.Yoghurt cultures are incubated at 42°C for 3-4 hrs with 1-2 % inoculum. It can be stored for one week without loss of activity.

12.2 Frozen, Deep Frozen and Frozen Concentrates

12.2.1 Frozen

The cultures are frozen to -20°C. Liquid starters (Mother and Feeder cultures) can be preserved for few months.

12.2.2 Deep freezing

Propagate cultures in continuously neutralized growth medium to optimize cell population. Cell mass is then separated and re-suspended in sterile growth medium and protective agent is added prior to packing and freezing. Preserved cultures are stored at -40 ° to -140°C.

The cryo-protective agents like sodium citrate, glycerol and sodiumβ-glycerophosphate etc., are used. In the absence of the cryo-protective agents there may be destruction of bacterial cells due to an increased concentration of electrolytes and other solutes inside the cell and in the outside suspending medium. The increased concentration of electrolytes and other solutes inside the cell results in denaturation of protein components and enzymes of bacterial cells, while the concentration of electrolytes outside the cell results in the dehydration of the protoplasm due to diffusion of the water through the cell wall membrane. Mechanical damage also may be caused as result of ice crystal formation.

12.2.3 Ultra low temperature freezing

This is carried outwith help of liquid nitrogen at -196°C using only sterile reconstituted SMP containing 16 % solids. Single strain inoculated at 2 % v/v, dispensed in 4 ml quantities in sterile polypropylene, screw top tubes and frozen at -196°C in liquid nitrogen. In this methods water molecules do not form large ice crystals. The biochemical processes inside the cell will cease i.e. the cell are at’ standstill’. These cultures can be stored for 2 years.

12.2.4 Freeze dried, vacuum dried and spray dried

12.2.5 Dried cultures

There are different methods of drying the cultures. They are:Spray drying, Vacuum drying, Freeze drying/ lyophilization, Concentrated freeze dried cultures (CFDC)

Advantages of dried cultures

• An alternative method for culture retention.

• To overcome the work involved in maintaining liquid stock culture

• It facilitates the dispatch of dried cultures by post without any loss in activity.

• Can be stored for a very long time

Disadvantages

• Dried starter contains less number of viable cells

• Require several subcultures before regaining maximum activity

The viability of preserved culture depends on

• Basic growth medium
• Presence of cryo-protective agents
• Rapid removal of metabolic compounds like lactic acid and carbonyl compounds
• Nature of suspending medium
• Conditions of freezing / drying
• Rate of thawing of deep frozen cultures
• Method of concentration referred as biomass concentration

Concentration of cell biomass is achieved by

a. Mechanical - by ultra centrifugation at 20,000 X g

1. Chemica l neutralization by removing or neutralizing acid. Usually ammonium hydroxide is recommended. Cell biomass is limited to 10¹⁰ cfu / ml L. bulgaricus withstands beyond 2 % lactic acid and S. thermophilus upto 1.5%.

2. Diffusion - fresh nutrients are permeated in and metabolic waste materials are diffused out through semi-permeable membranes. But some lactic acid may be retained which may limit the concentration of cell biomass.

12.2.6 Vacuum drying

Survival rate is very poor and is 1 -2 %. Several sub-cultures are required to obtain maximum activity.

Method of preparation

a. Mix the liquid culture with lactose

b. Neutralize the excessive acid with calcium carbonate

c. Concentrate the mixture partially by separation

d. Partially concentrated granules are vacuum dried

12.2.7 Spray drying

Evolved in Holland

The survival rate with mesophilic lactic acid bacteria is 10 % and with L. lactisssp. lactis biovar diacetylactis is 44 %. Survival rate can be improved by the addition of certain compounds like ascorbic acid and monosodium glutamate and propagation of cultures in buffered medium to increase viable cells per volume of sample or by neutralizing certain metabolites like lactic acid.

The activity of spray dried cultures can be retained for six months when stored at 21°C.

12.2.8 Freeze drying

This is also called as lyophilisation. This method enjoys widespread popularity because the survival rate is very high. In this method the cultures are dried in frozen state i.e.the organisms are frozen and then water is removed by “sublimation” i.e. from ice as vapour and thus preserving the structure and preventing the damage. Only a small quantity is required to inoculate the mother culture.

The process damages the bacterial cell membrane by the formation of ice crystals which can be minimized by the addition of cryo-protective agents to the cell suspension. The cells are also subjected to lethal effect due to the high presence of electrolytes as the ice crystals separate out. Both these processes can remove water from proteins and DNA and damage them. The protective solutes are of hydrogen bonding and or ionizing in nature which stabilize the cell membrane and prevent cellular injury during preservation

Starter culture is propagated in sterilized reconstituted SMP (16 %) at 2 %inoculation. The starters are held at 5°C for 18 hrs. Two to three ml is dispensed in sterilized glass vials. Vials are frozen at -40 °C for 2-3 hrs.They are freeze dried and vacuum sealed and stored at 5 °C. L.bulgaricus is sensitive for freeze drying.

1. Growth medium: Milk fortified with yeast extract and hydrolyzed protein. Neutralize the growth medium to pH range of 5-6 and propagate organisms at optimum growth temperature.

2. Cell biomass and suspending medium: Cell biomass has to have. 10¹⁰ cfu / ml and neutralize the suspending medium to 5-6 pH. Starter cultures are harvested towards the latter part of exponential phase with exception of L. bulgaricus which is in the early stages of stationary phase. The suspending medium for S. thermophilus is Skim milk + Sodium maleate and for L. bulgaricus it is Skim milk + lactose + Arginine Hcl solution and for Leuconostocs skim milk with glutamic acid

3. Freeze Drying: Freeze the cultures at -20°C to -30°C and dry at -10°C to 30°C to a final moisture of less than 3 %

4. Packaging: The cultures are vacuum packed as the presence of oxygen damages bacterial cells. Pack in glass ampoules, vials, laminated aluminum foil sachets.

5. Storage: 5-10°C

6. Reactivation: Follow the instructions of the manufactures. Re-hydrate at 20-25°C to prevent leakage of cellular ribonucleotides from damaged cells of L. bulgaricus. Others can vary.

12.2.9 Freeze dried concentrates

Traditionally 'bulk starter' in liquid form was used to inoculate the milk used in the manufacture of fermented products. Over the past few decades, the use of starter cell concentrates designated as either Direct Vat Set (DVS) or Direct Vat Inoculation (DVI) cultures have increasing being used, particularly in small plants, to replace bulk starter in cheese manufacture. Note that the terms DVI and DVS are used interchangeably although particular culture suppliers will tend to use only one term.

Starter concentrates used in DVI cultures are concentrated cell preparations containing in the order of 10¹¹-10¹³ cfu/g. They are available as frozen pellets or in freeze-dried granular form. Commercial DVS frozen cultures are in pellet form.

12.2.10 Production of starter concentrates

Under normal conditions starter growth in milk results in a cell concentration of about 10⁹CFU/ml. Growth of starters in milk is limited by a number of factors including the accumulation of lactic acid. Concentrates can be produced by neutralisation(traditional fermentation technology) or removal of the lactic acid (using diffusion culture), recovering the cells by centrifugation, not required if diffusion culture is used, and by maintaining starter activity by freeze drying or freezing. Freeze-dried concentrates can be stored for some months at 4 °C.Frozen concentrates are usually stored at -45°C or lower.

Higher cell densities(greater than 10¹⁰ cfu/g) can be obtained by harvesting the cells from the ferment or medium by centrifugation, to give a starter population of 10¹¹- 10¹² cfu/ml. Even higher cell densities can be obtained by freeze drying the 'sludge' obtained by centrifugation.

The starter bacterial cell population can be increased about 10-100 fold depending on the neutralizer used to neutralize the lactic acid formed. Both sodium hydroxide and ammonium hydroxide have been used and the use of the latter results in higher cell densities.

To maintain constant pH during the production of cell crops, the growth medium must be agitated.

12.2.11 Direct of vat cultures

Advantages

1. Convenience. The new generation of highly concentrated direct-vat inoculation cultures have the strength to go for inoculating much more milk with much less culture. The result is a narrowing of the cost-in-use gap between batch-set and direct-vat culture

2. Culture reliability. More reliable cultures can be obtained in direct-vat culture method

3. Improved daily performance and strain balance. Use of carefully selected bacterial strains along with innovative fermentation and blending technology allow cultures to provide a fast fermentation at a very low inoculation rate. A rotation of six cultures is available in convenient frozen pellet form.

4. Greater flexibility. The cultures can be chosen depending upon the requirement.

5. Better control of phage. Direct –vat cultures help to overcome phage issues, one of cheese makers' biggest nightmares. Phage viruses only pose a threat when the Direct –vat cultures are growing, unlike the greater window of time when batch-set cultures are being grown on site.

6. Possible improvement in quality. The direct-to-the-vat cultures can neutralize the lack of experience to a large degree if lot of experienced staff is not available. Training the staff regarding bacteriophage, sanitation, equipment operation, calibration etc., is a tough task but the direct-to-the-vat cultures streamline the educational curve”.

Disadvantages

1. Difficulties in providing liquid nitrogen facilities

2. Higher cost

3. Greater dependence on starter suppliers

4. Apportioning of responsibility in case of starter failure.