Table of Contents
- Microorganisms required for food fermentation may be added as pure cultures, mixed cultures, or, in rare cases, with no cultures at all if the appropriate microorganisms are present in sufficient numbers in the original raw material.
- In food fermentations for the production of sauerkraut, fermented pickles, and green olives, as well as in the processing of cocoa, coffee, poi, and citron, the original raw product contains sufficient quantities of the desired organisms, which will act in proper succession if favourable environmental conditions are maintained.
- Therefore, the addition of pure or mixed cultures of the organisms responsible for the fermentations has not been deemed required, although it is favourable in certain fermentations, such as pickling and green olives.
- In contrast, controlled “starter” cultures, whether pure or mixed, are typically employed in the production of fermented milks, some types of butter, and the majority of cheeses, as well as in the majority of other food fermentations, including bread, malt beverages, wines, distilled spirits, and vinegar.
General Principles of Culture Maintenance and Preparation
Selection of Cultures
- Cultures for food fermentations are chosen primarily based on their stability and their capacity to efficiently create the necessary products or modifications.
- These cultures may be established strains purchased from other laboratories, or they may be chosen following the testing of a large number of strains.
- Stability is a crucial quality; yields and rates of change must not be unstable. Some cultures, such as sporogenous yeasts, can be enhanced through breeding, but selection is the most prevalent strategy for improving strains.
- Selección de cultivos con caracteristicas deseadas puede ser realizada a partir de nuevas especies isoladas del medio ambiente, de especies existentes, o tras la alteration
- The improvement of plasmid transfer mechanisms in lactic acid bacteria will provide gene cloning or gene amplification of a highly desirable feature, such as lactic acid production.
- Due to the fact that the gene responsible for lactose fermentation in certain lactic streptococci is situated on plasmids, the trait is readily lost.
- It has been shown that it is possible to stabilise these industrially significant features. McKay and colleagues (Harlander et al., 1984) have cloned and integrated the lac + genes of S. lactis into S. sanguis.
- Using a vector plasmid and transformation, the lac + genes were inserted into the chromosome of the host cell.
- The lac + gene is significantly more stable when it is chromosomally integrated than when it is plasmid-borne.
Maintenance of Activity of Cultures
- Once a desirable culture has been established, it must be kept pure and alive. Typically, this objective is reached through periodic transfer of the culture to the appropriate culture media, incubation until the culture achieves the maximum stationary phase of development, and storage at temperatures low enough to prohibit further growth.
- Transferring an unstable culture too frequently may result in undesired changes to its traits.
- Stock cultures should be created for long-term storage of cultures without transfer. Such cultures tend to remain stable and serve as a source of culture in the event that the active culture deteriorates or disappears.
- Lyophilization (freeze-drying) and freezing in liquid nitrogen (196 degrees Celsius) are now commonly employed to create stock cultures, while a paraffin-oil seal is still sometimes applied to regular tube cultures.
- On agar plates containing 1 percent NaCl, bacterial cultures have been stored at room temperature for several months to years.
- A dry spore stock on sterilised soil can be used to store bacterial or fungal spores for extended durations.
Maintenance of Purity of Cultures
- To ensure the purity of cultures, they should be frequently purchased from a culture laboratory or their purity should be routinely examined.
- Methods for determining the purity of a culture differ depending on the type of culture being examined. Examination under the microscope will only reveal contamination if the contaminant has a distinct look and is abundant.
- A second technique involves plating the culture on an agar medium that will support the growth of contaminants but not the desired organism.
- Catalase in a culture of catalase-negative lactic acid bacteria can be used as an indicator of the presence of catalase-positive pollutants.
Preparation of Cultures
- Typically, mother culture is prepared daily from a prior mother culture and, originally, from stock culture.
- These mother cultures can be used to inoculate a larger volume of culture medium in order to produce the mass or bulk culture required for the fermentation process.
- Frequently, however, fermentation occurs on such a massive scale that many cultures of increasing size must be established between the mother culture and the bulk or mass culture.
- Culture makers strive to produce and maintain a culture that (1) contains only the desired microorganism(s), (2) is uniform in microbial numbers, proportions (if a mixed culture), and activity from day to day, (3) is active in producing the desired products, and (4) has adequate resistance to unfavourable conditions, if necessary, such as heat resistance, if it must withstand heating in a cheese curd.
- By standardising techniques of preparation and sterilisation of the culture media, inoculation, and incubation temperature and time, they attempt to preserve homogeneity.
- Depending on the intended usage, the culture will be grown to a specific stage of development.
- If they desire rapid and prompt growth, they employ a culture that is late in its logarithmic growth phase. If they desire more resilience to heat or other unfavourable conditions, they employ a culture that has just entered its maximum stationary phase.
- Exceptions apart, the temperature during incubation is typically close to the ideal temperature for the organism.
- Temperature and duration of incubation are frequently changed so that the culture is ready when required. Otherwise, it may need to be chilled to prevent further growth.
Activity of Culture
- The activity of a culture is measured by its growth rate and output of goods. If the mother or intermediate culture is adequate and the culture medium, incubation period, and temperature are optimal, the quality should be good.
- It is possible for cultures to deteriorate as a result of inappropriate handling and cultivation, frequent transfer over extended periods in an unsuitable culture media, selection, variation, or mutation, or bacteriophage attack.
- Sometimes, known mixes of pure cultures are generated by growing them together continuously or separately and combining them at the time of application.
- The so-called butter, or lactic, culture employed in the dairy business is an example of a mixture of multiple bacterial species and, at times, multiple strains of a single species.
- When multiple strains of the same species or distinct species are produced together, these organisms must be compatible, i.e., they must be able to coexist without causing the extinction of one another.
- It is challenging to maintain the proper balance of organism types in these mixed cultures. In some food preparations, the starters contain unknown mixes of microorganisms.
- Examples include the dough transferred from one batch of unique French bread to the next and the mixture of yeasts and bacteria transferred from the surface smear of one Limburger cheese to another.
With the exception of the propionic acid bacteria added to Swiss cheese, the majority of the bacterial cultures used as starters in dairy products, sausage, and bread are pure or mixed cultures of lactic acid bacteria. Bacteria that produce acetic acid are utilised in the production of vinegar, while other bacteria are used in the production of specific enzymes.
Lactic Acid Cultures
- Despite the fact that some dairy plants continue to effectively maintain their own cultures for years, the majority of operators receive new cultures on a periodic basis or use frozen, concentrated cultures generated by commercial culture laboratories.
- The bulk of commercially available cultures are now liquid-nitrogen-frozen culture concentrates made by cultivating cultures in a suitable medium, harvesting them, and then centrifuging them to concentrate them.
- Standardized for activity, the extracted cell pastes are then packaged in pull-top cans and immediately frozen in liquid nitrogen.
- It is conceivable to ship, distribute, and store frozen culture concentrates, and there are various benefits to doing so.
- Acquiring such cultures reduces the requirement for the maintenance and routine transfers associated with a company-maintained collection of cultures. Elimination of problems with culture contamination during transfer and periodic maintenance.
- It is possible to alternate the use of different strains of commercially available cultures to provide broader protection against bacteriophage infection.
- Many of these culture concentrate products contain such high cell counts that a 16-ounce container (containing 11 ounces of concentrate) can be used for direct inoculation of a cheese vat carrying up to 5,000 pounds of milk.
- This process would replace the previous method of preparing progressively larger bulk-starter inocula over the course of several days.
- Historically, the preparation of enough starter for a big cheese vat required several days of transferring the stock culture to larger and greater volumes.
- The most prevalent dairy starter is the lactic starter, which typically consists of a mixture of Streptococcus lactis subsp. lactis* and S. mesenteroides subsp. cremoris* for the production of lactic acid, and Leuconostoc* cremoris or Streptococcus lactis subsp. diacetlactis* for the production of flavour and aroma.
- Multiple strains of lactic streptococci with varying sensitivity to bacteriophage are typically included in the same culture concentrate as a precaution against phage problems.
- Generally, the lactic culture is incubated between 21.1 to 22.2 degrees Celsius, while 23.9 to 26.7 degrees Celsius has been used to make cheese starters.
- A culture is matured to the desired titratable acidity in accordance with its intended application.
- In the production of cultured buttermilk, yoghurt, and numerous forms of cheese, a mixed lactic culture is employed.
- Aroma bacteria play a crucial role in the flavour generation of cultured buttermilk. Typically, Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus are combined to form the yoghurt starter.
- The transfer and handling of mixed cultures must take into account the optimal temperature and duration of incubation for maintaining the intended mixed population and preventing the dominance of one strain or species over another.
- Using frozen concentrated cultures eliminates the majority of these issues. In addition, cultures from commercial culture laboratories allow for the purchase of appropriate strains that have already been determined and tested.
- The “sour” employed by rye bread bakers is a mixture of lactic acid bacteria that are typically produced in a mixed and impure culture in flour paste, dough, or another media.
- It is asserted that L. delbrueckii subsp. bulgaricus must develop in order to create adequate acid, and that heterofermentative lactics are beneficial from a flavour creation standpoint.
- Some breadmarkers inoculate their bread with pure cultures of Streptococcus lactis subsp. lactis, Leuconostoc spp., Lactobacillus plantarum, Lactobacillus casei, Lactobacillus brevis, Lactobacillus delbrueckii subsp. bulgaricus, and Streptococcus thermophillus. Other breadmakers include yeast as well.
- As starters for summer, Thuringer, and similar fermented sausages, Lactobacilli and Pediococcus acidilactici or P. pentosaceus cultures may be employed. Both cultures, dried cultures and culture concentrates frozen in liquid nitrogen, have been prepared.
- Propionibacterium freudenreichii cultures that have been spray-dried or lypohilized are added to the milk used to make Swiss cheese in order to enhance the flavour and promote the formation of eyes.
Cheese Smear Organisms
- The majority of cheesemakers inoculate the surfaces of smear-ripened cheese with microorganisms from previous cheeses, shelves, cloths, the brine tank, their hands, and other sources within the facility.
- Important smear micrococci, Brevibacterium linens, and film yeasts have been identified and utilised to inoculate cheese surfaces by washing them with pure or mixed cultures.
Acetic Acid Bacteria
- Pure cultures of Gluconobacter or Acetobacter are inefficient acetic acid producers.
- Therefore, impure mixed cultures are allowed to develop spontaneously, added via raw vinegar from a previous run, or transferred from a vinegar cask or generator during the production of vinegar.
The majority of industrially significant yeasts belong to the genus Saccharomyces, specifically the species S. cerevisiae. These yeasts that produce ascospores are easily bred for desirable traits. A yeast designed for a certain purpose can be optimised for that usage, but precautions must be taken to prevent unwanted modifications.
- Typically, the S. cerevisiae strains used to produce baker’s yeast are single-cell isolates that have been specifically chosen for this purpose.
- They should produce a high yield of viable cells in the mash or medium used for their cultivation, be stable, retain their viability in the cake or dried form for an extended period of time, and produce carbon dioxide quickly in bread dough when employed as leavening agents.
- Similar to other large-scale cultures, this culture is constructed from the original mother culture through multiple intermediary cultures of increasing size to the “seed” culture.
- The cells from the seed-culture tank are concentrated into a “cream” by centrifugation, and this dense suspension is introduced to the enormous volume of mash in which the yeast will be cultivated, along with 3 to 5 pound of yeast per 100 gallons of medium.
- The most common medium for the growth of cultures and the production of bakers’ yeast is a cane or beet molasses-mineral-salts mash containing molasses, nitrogen foods in the form of ammonium salts, urea, malt sprouts, etc., inorganic salts as phosphates and other mineral salts, and accessory growth substances in the form of vegetable, grain, or yeast extracts, or small amounts of vitamin precursors or
- Adjust the pH to between 4.3 and 4.5, and the incubation temperature to roughly 30 degrees Celsius. During yeast development, the medium is rapidly aerated, and molasses is added progressively to maintain a sugar concentration of between 0.5 and 1.5 percent.
- After four or five cycles of budding, the yeast is centrifuged into a “cream,” which is then pressed through a filter press to remove superfluous liquid. After incorporating modest amounts of vegetable oils into the yeast mass, cakes of varying sizes are created.
- Currently, active dry yeast is produced by dehydrating yeast cells to less than 8 percent moisture. The cells are cultivated specifically for this purpose and carefully dried at low temperatures so that the majority of cells survive and preserve their ability to leaven dough for several months.
- Additionally, baker’s yeast can be produced from grain mashes, waste sulfite liquor from paper mills, and wood hydrolysate.
Yeasts for Malt Beverages
- Yeasts for malt drinks may be stored in pure culture in brewery laboratories or procured from specialised laboratories as needed.
- The strain chosen is one suited to the product to be brewed, a special bottom yeast for beer, typically a top yeast for ale, but occasionally a bottom yeast, and a top yeast for stout and porter.
- The most common yeasts are strains of Saccharomyces cerevisiae, whereas S. uvarum is the least common (carlsbergensis).
- When yeast is grown from a pure culture, it must be grown in wort from a laboratory culture to a large seed culture or “pitching” culture. However, in practise, pitching yeast is almost usually yeast recovered from a prior fermentation.
- Concentrated yeast that may or may not have been washed. Clearly, such a yeast culture will always be contaminated with other species, typically bacteria and wild yeasts that accumulate during subsequent fermentations and recoveries.
- Even while most pollutants can thrive in the yeast culture, they are hindered by the hops and low pH in the wort and do not significantly harm the malt beverage.
- For the production of wine, a strain of Saccharomyces cerevisiae var. ellipsoideus suitable to the production of the particular type of wine is chosen.
- The extensively utilised Burgundy, Tokay, and champagne cultures are well-known kinds.
- The cultures are developed and augmented in must (grape or other fruit juice) that will be used for the primary fermentation.
- Typically, distillers’ yeast is a high-alcohol-yielding strain of S. cerevisiae var. ellipsoideus that has been modified to grow in the intended medium or mash.
- The medium or mash is typically malted grain, such as corn or rye for whiskey, molasses for rum, or fruit juices or mashes for brandy.
- The liquors are produced by distilling the fermenting mashes.
- Mold stock cultures are typically grown on slants of a suitable agar medium, such as malt-extract agar, and may be maintained in the spore stage for extended durations through lyophilization (freeze drying) or as soil stocks.
- There are various ways to prepare spore or mycelial cultures for usage on a plant scale. These consist of
- Growth on the surface of a liquid or agar medium in a flask or other container.
- In shallow layers in trays, surface growth on media.
- growth on loose, moist wheat bran that may be acidified or to which liquid nutrients, such as corn-steep liquor, may be supplied.
- Bread or crackers that have been sterilised and soaked prior to growing.
- The consequence of submerged development in an aerated liquid medium is often pellets of mycelium, with or without spores.
- Mold spores are collected in a variety of methods, depending on the production method.
- They may be cleaned or extracted from dry surfaces, left in dry materials that have been ground or powdered, or for ease of usage, integrated into a dry powder, such as flour.
- The pellets are, of course, utilised as pellets. Spores of Penicillium roqueforti for blue cheeses, such as Roquefort, Stilton, Gorgonzola, etc., are typically cultured on cubes of sterilised, moistened, and typically acidic bread; whole wheat or bread made with a particular formula may also be used.
- After mould sporulation is complete, bread and culture are dried, pulverised, and typically packaged in cans. P. camemberti spores are produced by cultivating the fungus on sterile, damp crackers.
- For the surface inoculation of the Camembert, Brie, or similar cheese, a spore suspension is created.
- Mold starters used as inoculum in industrial submerged fermentations are often formed as pellets or masses of mycelium during submerged growth while the culture is vigorously shaken.
- When surface growth is sought on a liquid, agar, or bran medium, mould spores produced by one of the aforementioned ways are typically used as the inoculant.
- The koji, or starter, for soy sauce, which is discussed in the following chapter, is often a mixture of cultures carried over from a previous batch, although pure cultures of Aspergillus oryzae, yeast, and Lactobacillus delbrueckii have been employed.
- The mould culture is cultured on sterilised, cooked rice.