Starter Lactic Cultures: Fermentation, Management and Preparation | Industrial Microbiology

In this article we will discuss about the starter lactic cultures:- 1. What is Starter Lactic Culture 2. Biochemical Basis of Culturing Dairy Products Using Starters 3. Management and Preparation 4. Continuous Starter Production 5. Starter Defects 6. Shelf Life Extension by Lactic Cultures. Learn about:- 1. Starter Culture in Dairy Industry, 2. Starter Culture in Fermentation and 3. Starter Culture in Cheese.

Contents:

1. What is Starter Lactic Culture
2. Biochemical Basis of Culturing Dairy Products
3. Management and Preparation of Starters
4. Continuous Starter Production
5. Starter Defects
6. Shelf Life Extension by Lactic Cultures

1. What is Starter Lactic Culture?:

Milk is the normal habitat of a number of lactic acid bacteria which may cause spontaneous souring. For sophisticated control, modern industrial processes utilize specially prepared lactic acid bacteria as starter cultures, or “starters,” in the manufacture of fermented dairy products.

A wealth of information on the starter cultures is available in the literature. A starter consists of harmless microorganisms which, upon culturing in milk or milk-based mixes, impart desirable and predictable characteristics of flavor and texture attributable to a certain fermented milk product.

A single-strain culture contains an individual strain of a bacterial species while a mixed/multi-strain culture consists of a mixture of more than one strain or species. In the United States, Canada, and the Netherlands, a starter generally consists of mixed strains. A multi-strain starter is consid­ered to have an advantage over a single strain starter since fermentation will continue in the presence of a phage which specifically attacks one strain only.

However, in mixed strain starters, a single strain may domi­nate at the expense of other constituent strains. A considerable number of plants are now using two mixed-strain starters. In Australia and New Zealand, the preferred technology is to grow two single starters individually and to mix them prior to culturing of cheese vats. The advan­tage of this technique is that an individual strain, preselected for its per­formance and resistance to antibiotics and phage, is used for ensuring greater predictability and uniformity of culture effects.

For distribution of starters an earlier method involved shipping them as a liquid subculture. The liquid cultures are generally no longer distributed in commercial practice but, in rare cases, may be useful in distribution of cultures from a central laboratory to an opening plant. To prepare a liquid culture, the organisms are propagated in a suitable medium such as milk or whey and maintained in an active condition by periodic transfers.

In gener­al, a liquid culture contains about 10⁹ organisms/ml of the starter. In addition to the continuous care required to maintain liquid cultures, it has been observed that repeated transfers may cause the culture to lose some of its critical characteristics. This problem is minimized by freeze-drying the cultures grown in milk and distributing the lyophilized culture in small vials.

The freeze-dried cultures can be stored at room temperature for several years but the degree of viability of the organisms is very low. Reactivation of the lyophilized culture is necessary for proper performance. A study on the preparation of freeze-dried concentrates of the starters for direct vat inoculation has been reported by Sandine (1977).

The storage stability appears to be enhanced by β-glycerol phosphate. Kilara et al. (1976) studied the effect of cryoprotective agents on freeze-drying and storage of lactic cultures. After 48 weeks, casitone yielded 15% survival of Lactobacillus bulgaricus and monosodium glutamate gave 36% survival of Lactobacillus acidophilus. Malt extract was found to give 30 and 22% survival for Streptococcus thermophilus and Streptococcus lactis, respec­tively.

Presently, the most common method for distribution of lactic cultures is in the form of frozen concentrates. The cultures are grown under optimum conditions in a fermentor, concentrated by centrifugation, and suspended in a suitable medium for maximum protection during freeze/thaw cycle and flash frozen in liquid nitrogen at -196°C. Culture concentrates contain approximately 10¹¹ organisms/ml.

The activity of a starter, frozen at -196°C, is generally very satisfactory if it is thawed to 30°C and imme­diately inoculated into fresh substrate, High survival rates (95% viability) for starters can be achieved by this method. The use of frozen concentrated cultures in cultured dairy plants has eliminated the need for routine main­tenance of a culture collection at the plant level.

The culture concentrates are standardized for activity by the culture manufacturer. The concentrates may be designed for use in bulk starter preparation or for direct seeding in the culture vat. This development has virtually eliminated major incidents of contamination by bacteriophage or other undesirable organisms.

According to Bergey’s Manual, lactic acid bacteria generally used in fermented milks constitute the following two groups:

In general, two distinct types of fermentation processes are involved. All the products result from lactic acid fermentations.

In addition, kefir and koumiss Utilize alcoholic fermentations by lactose-fermenting yeasts which produce up to 3% alcohol and CO₂ to impart effervescence. For buttermilk, sour cream, and cream cheese, it is customary to use a heterofermentative leuconostoc or Streptococcus lactis subsp. diacetylactis to generate flavor compounds (e.g., diacetyl) typical of such products.

These properties are helpful in the utilization and control of the various fermentation processes in the industry. The starters are graded according to their acid production, rate of growth, phage and antibiotic resistance, and ability to develop typical flavor and texture.

The bacteria commonly used in cultured dairy foods are- for acid production, Lactobacillus acidophilus, Streptococcus thermophilus, Streptococcus lactis, and Streptococcus cremoris; for acid produc­tion and flavor development, Lactobacillus bulgaricus, Streptococcus lactis subsp, diacetylactis and for flavor production, Leuconostoc cremoris.

In some instances Lactobacillus lactis, Lactobacillus helveticus, Leuconostoc dextranicum, Streptococcus durans, and Streptococcus faecalis are used for acid and flavor production and Propionibacterium shermanii is used for flavor development. A reasonable degree of versatility in acid and flavor production may be achieved by combining different strains and species of these organisms.

The starters are generally purchased from commercial sources specializ­ing in their production and marketing. Among the major culture suppliers are- Chr. Hansen’s Laboratory, Milwaukee, WI; Marshall Div. of Miles Laboratories, Madison, WI; Microlife Technics, Sarasota, FL; Dairyland Food Labs., Waukesha, WI; Vivolac Cultures, Indianapolis, IN; Wiesby Laboratorium, Niebull, Germany; and Flora Donica, Odesse, Denmark.

Pure cultures may be obtained from the American Type Culture Collection, Rockville, MD; Northern Regional Labs., Peoria, IL; the National Dairy Collection, National Institute of Research for Dairying, Reading, England; and the Institute for Fermentations, Osaka, Japan. Dairy research centers in Australia (Central Scientific and Industrial Research Organization), New Zealand (Dairy Research Institute), Holland (The Netherlands Instituut Voor Zuivelonderzoek), France (Jouy-en-Josas), and Switzerland (Lie- befeld) maintain a good supply of lactic cultures.

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2. Biochemical Basis of Culturing Dairy Products Using Starters:

The production of lactic acid, acetic acid, CO₂, diacetyl, and acetaldehyde from lactose and citric acid is of fundamental importance to the growth of lactic cultures and generation of characteristic flavor in products. The fermentation reactions take place in series, em­ploying both homofermentative and heterofermentative systems.

Homofermentative lacto- bacilli and streptococci hydrolyze lactose into glucose and galactose. The formation of pyruvic acid follows the Embden-Meyerhof glycolytic path­way, hexose monophospate shunt pathway, Leloir pathway, and D-tagatose-6-phosphate pathway.

Gilliland et al. (1972) reported stimulation of lactic streptococci by the hydrolysis of lactose in milk to glucose and galactose by β-galactosidase. Using lactose and citric acid as primary substrates, significant quantities of typical me­tabolites are produced and accumulated in the fermented milks.

Diacetyl imparts characteristic flavor to cultured butter, buttermilk, and sour cream. The production of diacetyl may be enhanced by environmental manipulation and by the selection of suitable strains of bacteria producing more diacetyl in preference to acetoin. Production of diacetyl generally increases below pH 5.5.

The fermentation profile of homofermentative lactobacilli is dependent upon the level of substrate and the degree of aeration Limiting concentrations of glucose and galactose result in production of lactate, acetate, and CO₂. Under aerobic conditions, cultures produce pri­marily acetic acid. However, many manufacturers enhance the acid-produc­ing activity of starters by removal of oxygen from milk using a vacuum treatment.

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3. Management and Preparation of Starters:

The advent and industry-wide acceptance of frozen culture concentrates has simplified the management of cultures in most cultured dairy plants in the United States. However, working knowledge and employee training in lactic cultures are still advantageous in handling starters at the plant level In countries where frozen culture concentrates are not yet fully developed or in case certain plants use proprietary strains and cultures in their plants it is essential to develop and maintain appropriate microbiological expertise in the propagation, maintenance, and control of lactic starter cultures.

The starter is the most crucial component in the production of high quality fermented milks. Culture propagation should be conducted in a specified, secluded area of the plant where access of personnel is restricted An effective sanitation program coupled with filtered air and positive pressure m the culture area, and preferably all the manufacturing areas including the packaging room, should significantly reduce the airborne contamination. Consequently, culture failure due to phage may be con­trolled, and extended shelf life of the product may be attained.

The media for culture propagation are generally composed of liquid skim milk or blends of cheese whey and nonfat dry milk dispersed in water so as to contain 9-10% milk solids. An improved whey-based phage inhibitory medium is available. Water, nonfat dry milk whey, and other media ingredients must be free from substances inhibitory to the growth of the starter.

Such inhibitory substances include sanitizing chemicals such as chlorine, iodine, and quaternary ammonium compounds as well as antibiotics and phages. Special media for optimum culture activ­ity and phage resistance are available from commercial culture companies The media generally contain demineralized whey, nonfat dry milk phos­phate, citrate, and growth factors present in yeast extracts.

Phosphate acts as a sequestrant of Ca⁺ and thereby inhibits Ca^{+ +} dependent phage growth. Citrate provides a substrate for production of diacetyl and, along with phosphate, contributes to the buffering capacity. The powdered media are generally dispersed in water as such or blended with an equal weight of nonfat dry milk to attain 10-12% solids. Alternately, the media may be dispersed in liquid skim milk.

Heat treatment is necessary to destroy the contaminants in the medium and to alleviate unnecessary competition to the growth of a desirable lactic culture. In addition, heating the medium produces desirable nutrients by heat-induced reactions in milk constituents.

At present, cultures are purchased on a regular basis from commercial suppliers. They may be lyophilized or frozen concentrates shipped in liquid nitrogen or dry ice. For extended storage, the culture concentrate cans must be stored in liquid nitrogen. However, for relatively short storage periods of 4-6 weeks, the cultures may be stored in special freezers (at -40°C).

The use of freezers offers an economical alternative if the turnover of cultures at the plant level is high and proper care in culture can rotation is taken. The culture concentrates may be designed for bulk starter prepara­tion or for direct inoculation into product mixes. The use of frozen culture concentrates eliminates the preparation of mother cultures and intermedi­ate cultures.

The culture growth conditions are applicable to mixed strains of Streptococcus lactis, S. cremoris, S. lactis subsp. diacetylactis, and Leuconostoc cremoris. The procedure involves the use of nonfat dry milk as well as special media developed by several manufacturers for pro­ducing bulk starters.

Control of acid and flavor development in lactic starter cultures may be achieved by understanding their growth characteristics. By modifying the inoculation rate, incubation temperature, and time, it is possible to direct the fermentation, in a limited way, to fit the plant schedules. Care should be taken to preserve the balance of strains and organisms in the culture so that a symbiotic relationship is maintained.

Yogurt culture consists of two lactose-fermenting organisms, Lactobacil­lus bulgaricus (rod) and Streptococcus thermophilus (coccus). Culturing the two organisms together results in a symbiotic relationship since the growth rate and acid production by each organism are greater when grown together than in a single culture. Optimum growth temperatures for the rod and coccus are 45°C and 40°C, respectively.

Depending upon incubation tem­perature, a differential in the ratio of rod to coccus would be evident. For full yogurt flavor development, a ratio of 1:1 is generally accepted as ideal. Upon repeated transfers, this ratio tends to change, depending upon the incubation temperature.

A reasonable success in rectifying the balance has been achieved by varying the rate of inoculum, incubation time and tem­perature, acidity level in milk, and heat treatment of milk. Using a 2% inoculum and incubation at 44°C for 2.5 hr, proper balance of rod and coccus can be maintained in a yogurt culture. If the ratio is not 1:1, the streptococci may be increased by lowering incubation time or temperature. Conversely, the lactobacilli population may be encouraged by higher incubation time or temperature.

The yogurt culture may be obtained as a lyophilized culture or more commonly as a frozen concentrate. For the preparation of bulk starter, the procedures may be used with the exception of an incuba­tion temperature of 44°C. Whole milk medium may be autoclaved at 121°C for 10 min, cooled to 44°C, inoculated with 2% inoculum, and incubated at 44°C until a pH of 4.6-4.7 is attained.

Upon repeated transfers, optimum activity of the culture is obtained when the desired acidity is produced in 2.5 hr. Mother cultures are propagated daily for a period of about 2-3 weeks after which a fresh lyophilized cultures is activated. Different strains of frozen culture concentrates are rotated on a daily basis. General micro­scopic examination of the culture is recommended to ensure correct balance between rod and coccus.

New developments in starter technology have been summarized by Law­rence etal. (1976), Gilliland (1977), and Sandine (1977). For the preparation of concentrated cultures, batch processes are favored. The literature reviews indicate that a pH of 6.0-6.3 is optimum for maximum starter cell production and the pH should be maintained by neutralization, preferably with NH₄OH.

The addition of active catalase stimulates acid production by lactic culture during fermentation. The cata­lase destroys the inhibitor hydrogen peroxide elaborated by the culture during its growth. The frozen cultures display maximum viability and activity upon thawing when they have been stored at a temperature of -196°C.

However, Stadhouders et al. (1971) showed that storage at -37°C is equally effective if the culture contains 7.5% lactose prior to freezing The survival of the lactic culture during freezing at – 17°C has been shown to be related to the fatty acid composition and glucose level in the cells The amount of cellular capsular material also appears to be involved.

The application of genetic manipulation of lactic cultures in the technol­ogy of fermented dairy products is of particular interest in the future. Recent work of McKay and Baldwin (1975) suggests that the genes control­ling proteolytic characteristics are located in plasmids. Lawrence et al (1976) have reviewed the studies relative to lysogeny, transduction, plasmid analysis, modification-restriction system, and phage carrying state of lactic streptococci.

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4. Continuous Starter Production:

Lloyd (1971) and Kosikowski (1977) have reviewed the developments in this area. The design and use of equipment for continuous starter produc­tion provides a potential for mechanization and automation in the cheese and cultured dairy products industry.

The concept of continuous starter production is based upon the basic method for continuous propagation of microorganisms. The equipment consists of a large fermentation vessel with several sealable ports. Also, a provision is made for maintaining constant conditions of temperature, pH oxygen tension, and pressure.

Milk or a suitable medium is introduced from the inlet port and concomitantly the starter is pumped out. In this fashion the system functions as a feed and bleeds operation. Adequate care is taken to ensure a balance between the output of bacteria in their growth phase and the inflow of nutrients. The acidity is maintained at a level low enough to prevent injury to the bacterial cells.

A continuous culture system pro­vides microorganisms in an exponential phase of growth in a constant stream using minimal substrate concentration. Accordingly, this system is valuable in studying regulation of synthesis, mutant selection, ecological behavior, and catabolism of the limiting substrates. In a chemostat, the flow rate (bleed rate) is coordinated with the growth rate of the culture. In a turbidostat, the culture density is measured by a turbidity-sensing device which regulates the flow rate.

Several workers have reported continuous systems for lactic starter growth. Wilkowske and Pouts (1958) found optimum rate of starter produc­tion at pH 5.3. Ashton et al. (1959) produced starter for direct inoculation into a cheese vat. Berridge (1966) devised a continuous system of Strepto­coccus lactis subsp. diacetylactis fermentation with the pH controlled by the flow rate.

More acid was formed at pH 6.0 than at a lower pH. Continuous fermentation systems for milk products with an output of 500 liters/hr have been reported. Robertson (1966) and Lattey (1968) explored the possibility of continuous starter systems at pH 5.0-6.3, and preservation of the culture by freezing at -196°C and storage at -75°C.

Bulk frozen concentrates have been prepared using continuous fermentation in a whey medium containing yeast extract. Starter concentrates for direct vat inoculation have been demon­strated by continuous culturing of Streptococcus lactis C2 in trypsin- digested whey fortified with yeast extract. Pepsin-hydrolyzed whey medium reportedly gave better results. Lewis (1967) reported maintenance of a yogurt culture continuously for a month in a chemostat. Details concerning a satisfactory pilot plant for continuous production of Streptococcus lactis have been described.

The challenge in continuous culture technology is threefold. First, con­tamination with phage and other undesirable organisms has to be avoided during lengthy production runs. Also, the formation of undesirable mutants must be prevented. Secondly, problems in biological equilibrium among various strains or symbiotic relationships must be resolved in order to command industrial application. Thirdly, the development of continuous systems must be technologically and economically viable for cultured dairy plants to switch from frozen culture concentrates.

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5. Starter Defects:

During continuous growth the starter organisms may remain active and preserve their characteristics for some time. However, they may lose their activity rapidly depending on the compatibility of the species and strains. Also, activity is lost or changed due to the physical environment. In any case, change from the normal fermentation pattern is considered a defect.

The common defects are:

1. Insufficient Acid Development:

This is one of the common defects in lactic cultures. Kosikowski (1977) defines a slow starter in terms of 1 ml culture which, upon inoculation into 10 ml of antimetabolite-free, heat- treated milk, produces less than 0.7% titratable acidity in 4 hr at 35°C.

Factors contributing to a slow starter are:

(a) Composition of Milk:

Certain raw milks exert an inhibitory effect on many lactic starters, and this is attributed to various natural inhibitors including lactenins, lactoperoxidase, agglutenins, and lysozyme. These inhibitors are present in all milk and show considerable variations with breed and season.

All these factors are heat-labile and their inhibitory property is arrested progressively on heating. When milk is pasteurized at 72°C for 16 sec or autoclaved for 15 min, the natural inhibi­tors are completely destroyed. Further, the growth of starter cultures is stimulated in heated or autoclaved milk due to partial hydrolysis of casein, liberation of sulfhydryl groups, and formation of formate from lactose.

Rapid acid production by lactic acid bacteria is observed in milk heated at 90°C for 1 hr, or 116°C for 15 min, or 121°C for 10 min. Autoclaving treatments are generally avoided for intermediate and bulk starter prepa­ration because of the introduction of undesirable caramelized color and flavor in milk. However, flavor producing strains of Leuconostoc cremoris grow better in milk sterilized at 121°C for 15 min.

Recent trends in ultra-heat or ultra-high temperature treatment (UHT) of milk as a means of extending shelf life appear to have interesting implica­tions for the cultured dairy product industry. Stone et al. (1975) demon­strated stimulation in growth of lactic starters in UHT milk. It appears that UHT milk is a better medium for culture growth than milk processed by batch or short-time pasteurization procedures.

Milk from mastitis-infected animals generally does not support the growth of lactic cultures. This effect is ascribed to the infection-induced changes in chemical composition of milk. For example, mastitis milk con­tains lower concentrations of lactose and unhydrolyzed protein and higher chloride content and a higher pH than normal milk. Furthermore, a high leucocyte count in mastitis milk inhibits bacterial growth by phagocytic action. Heat treatment restores the culture growth in mastitis milk.

Colostrum and late lactation milk contain nonspecific agglutenins which clump and precipitate sensitive strains of the starter. The agglutenins may possibly retard the rate of acid production by interfering with the transport of lactose and other nutrients.

Seasonal variation in the solids-not-fat fraction of milk affects the growth and the balance of strains in culture. Generally, a higher solids-not-fat level in milk favors the growth of lactic cultures. The ratio of leuconostocs to streptococci may be maintained by the addition of 0.02 M Mn⁺⁺/ml of the medium.

(b) Contaminating Microorganisms:

Prior degradation of milk constit­uents by contaminants affects the growth of lactic organisms. Pre-culturing of milk with psychrotrophic organisms enhanced acid production by Strep­tococcus thermophilus, Lactobacillus bulgaricus, Streptococcus lactis, and Streptococcus cremoris. However, careful screening of milk for psychrotrophs is necessary for quality flavor produc­tion by lactic cultures.

(c) Antibiotics and Chemicals:

Various antibiotics gain entry into milk during antibiotic treatment of mastitis, and these inhibit acid production by bacteriostatic action, depending on the type of starter and the kind and amount of antibiotic involved. Concentrations as low as 0.005-0.05 Inter­national Units of penicillin, aureomycin, terramycin, or streptomycin per ml of milk are high enough to produce partial or full inhibition of the starter.

To overcome this difficulty many workers have suggested use of antibiotic resistant starter cultures and the use of additional materials like pancreatic extract or enzymes like penicillinase. However, a practical con­trol is exercised by routine examination of milk and by avoiding use of milk showing a positive antibiotic test.

In this regard, a rapid technique for antibiotic detection is particularly useful. The technique is based upon the inhibition of acid production by the assay organism Bacillus stearothermophilus var. calidolactis in the presence of extremely low antibiotic levels. The results are available in 2.5 hr.

Many sanitizing chemicals like quaternary ammonium compounds and chlorine compounds retard acid development by starter cultures. One to 5 parts per million of these sanitizing compounds are bactericidal to lactic cultures. Consequently, it is important to exert care and control in the use of sanitizers in the plant. Fatty acids (C-10 to C-16) also inhibit starters.

These fatty acids may be due to partial hydrolysis of milk by lipases or they may be produced by lipolytic organisms. The fatty acids, particularly lauric, caprylic, and capric, lower the surface tension of milk to less than 40 dynes/cm. The inhibition of lactic cultures by free fatty acids is apparently related to the surface activity of the growth medium.

Avoiding the use of rancid milk is important not only from the standpoint of culture growth but more significantly because it would impart an objec­tionable flavor to the starter and the cultured dairy products derived there from.

Environmental pollutants such as telodrin, dieldrin, lindane, DDT, PCB, and PBB may be found occasionally in milk. However, the insecticides have little or no effect on the growth or fermentation ability of lactic cultures. Murata et al. (1977) were able to culture milk containing PBB below 0.3 ppm on a fat basis. More work is needed to assess the impact of these pollutants on starters.

(d) Change in Fermentation Behavior:

After continuous use, the start­ers may change their fermentation activity and consequently produce lower amounts of lactic acid. This is attributed to genetic changes brought about by environmental factors.

When lyophilized or refrigerated organisms are used to prepare starters there may be a marked decrease in acid production. Some workers have suggested the use of various chemicals like glycerine, azide, and sucrose to restore original acid-producing capacity. Acid production is stimulated by adding 0.25% pancreatic extract. This stimulation is attributed to the various nucleic acid bases present in the added extract.

Strain dominance in mixed cultures, causing changes m the behavior of the composite cultures, is well documented. Results show that Streptococcus lactis subsp. diacetylactis tends to dominate Strep­tococcus lactis or Streptococcus cremoris. Different strains of Streptococcus lactis display major differences in domination during associative growth with Streptococcus cremoris.

(e) Phage Action:

Attack by bacteriophages is an important cause of slow acid production by lactic cultures. When the phage has reached a maximum level, all sensitive bacterial cells are infected and lysed within 30-40 min. When lysis occurs, acid production by the affected culture stops unless some resistant bacteria are present to carry on fermentation Phages, strain-specific viruses, consist of a head (70 nm wide) and a tail (200 nm x 30 nm) The phage attacks streptococci as well as lactobacilli by attachment of the tail to the bacterial cell followed by injection of DISA from the phage head into the cell.

This is followed by synthesis of new phage particles and cell lysis which releases up to 200 new phage particles to carry on further attack and lysis of the bacteria. If at least 50% of the fast acid-producing bacteria are phage resistant, a phage attack is not discernible.

Phage control is affected in cultured milk plants by using 200-300 ppm chlorine on processing equipment and by fogging the culture rooms with 500-1000 ppm of chlorine. Heat treatment of milk (75°C for 30 min or 80°C for 20 sec) is considered adequate to inactivate various phages which attack lactic acid bacteria. By using combinations of proper procedures such as sanitation, culture selection, and culture rotation, the probability of trouble with phage can be minimized. Use of phage-resistant and multiple-strain cultures is generally preferred.

The role of calcium in infection of starters by phage has been determined. Since phages which attack lactic cultures require calcium for their activity, a medium with reduced calcium may be used to propagate starters. Calcium can be removed by ion exchange or by treating milk with various polyphosphates, phosphates, ammonium oxalate, and sodium salts of EDTA. Ammonium oxalate was rated better than other additives for removal of calcium but phosphates are generally used because of their general acceptance as food additives.

In certain instances, acidity may be too high in relation to product standards. This problem is encountered in yogurt production. High acidity is usually associated with high incubation temperature, long incubation period, or excessive inoculum.

2. Insufficient or Abnormal Flavor Development:

Adequate production of lactic acid is essential for lowering the pH to a level where diacetyl and other compounds are formed in sufficient quantity. For good flavor any factor interfering with proper acid development will retard or prevent adequate flavor development.

The culture may be incapable of producing adequate amounts of flavor due to a change in fermentation pattern induced by oxygen tension or due to a change in the balance of various bacterial cultures. Flavor can be improved by adding lactose, citric acid, and oxalacetate. The flavor can be enhanced by acidification of milk and inoculating with culture concenctrates of Leuconostoc cremoris.

The common flavor defects are maltiness, metallic flavor, methyl sulfide flavor, green flavor, fishy flavor, and fruity flavor. Maltiness is produced by the growth of Streptococcus lactis var. maltigenes. This organism is capable of surviving pasteurization. The malty flavor is caused by the production of 2-methyl propanal, 3-methyl butanal, 2-methyl propanol, and 3-methyl butanol.

Metallic or puckery flavor is chiefly due to metallic contamination of the starter. Sometimes, overgrowth of leuconostocs also contributes to this development,

Green flavor in buttermilk is generally due to the production and accu­mulation of acetaldehyde in excessive amounts. In normal fermenting cultures, the acetaldehyde formed is metabolized by homo-fermenters to produce ethanol. In abnormal fermentations, hetero-fermenters produce acetaldehyde in excessive amounts which homo-fermenters are unable to metabolize.

A diacetyl acetaldehyde ratio of 3:1 to 5:1 produces good buttermilk and sour cream flavor. It the ratio is 0.4:1, a green flavor is experienced by the taster, Harsh or yogurt flavor results when the ratio is 13:1. Acetal­dehyde is an essential component of yogurt flavor Approxi­mately 25 ppm acetaldehyde is produced by symbiotic activity of a yogurt culture.

Fruity flavor is observed during some abnormal fermentations and is produced when excess acetaldehyde is converted to esters, ethyl hexanoate and ethyl butyrate.

Fishy flavor is associated with the liberation of trimethylamine and related compounds by bacteria. Also, this flavor may be due to nonbacterial lipid oxidation.

Methyl sulfide flavor is produced by Aerobacter aerogenes which may be a contaminant of the starter. This flavor is also referred to as cowy or feedy or both.

3. Ropiness and Gassiness:

Ropiness or slimy milk is due to the change m the character of lactic streptococci or due to the dominance of the ropy strain of Leuconostoc mesenteroides. Psychrotrophic contaminants like AL- caiigenes visolactis, Aerobacter aerogenes, and pseudomonads are often responsible for this defect.

Gassiness is due to the accumulation of gas during fermentation. A starter containing Streptococcus lactis subsp. diacetylactis may liberate large amounts of CO₂ gas. Among contaminants, organisms of the Escnerichia-Enterobacter group are chiefly responsible for this effect.

4. Bitterness:

This defect is due to limited proteolytic activity of some starter strains, and is commonly observed with Cheddar cheese cultures. Also, it can be attributed to the presence of proteolytic bacteria in the starter culture, Streptococcus liquefaciens and some sporeformers which survive normal heat treatments of the milk are the usual causes.

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6. Shelf Life Extension by Lactic Cultures:

Cultured milk products have been reported to contain antibacterial prin­ciples which apparently contribute to their increase in shelf life and control of food-borne pathogens. Evidence has been mounting on the occurrence of antibiotic materials in fermented milk products.

Lactic acid, acetic acid, and hydrogen peroxide are additional factors inhibitory to spoilage organisms. Pseudomonas fragi is inhibited by a volatile fraction from a medium cultured with Streptococcus diacetylactis. Similarly, Salmonella typhimurium and Salmonella gallinarum are inhibited by Leuconostoc cremoris. Several lactobacilli have been reported to repress the growth of Staphylococcus aureus and several species of Pseudomonas.

Actively growing starters, particularly Streptococcus lactis, inhibit the formation of staphylococcal enterotoxin A. The occurrence of natural benzoic acid (up to 50 ppm) in cultured dairy products has been substantiated.

Filtrates of skim milk cultured with Leuconostoc cremoris inhibited, to a varying degree, Staphylococcus aureus, Pseudomonas fluorescens, Escher­ichia coli, Pseudomonas fragi, and Enterobacter aerogenes. Daly et al. (1972) determined the effect of Streptococcus diacetyIactis on the growth of various organisms in milk.

Inhibition ranged from 70 to 99.9% for Pseudomonas fluorescens, P. fragi, P. viscosa, P. aeruginosa Clostridium perfringens, Escherichia coli, Salmonella tennessee, and Vibrio parahemolyticus. Evidently, the inhibitory effect is caused by an antimicro­bial metabolite as well as low pH.

Branen etal. (1975) found Streptococcus diacetylactis and Leuconostoc cremoris to inhibit Pseudomonas putrefaciens, P. fragi, and P. fluorescens, but wide variations were observed in different strains of the lactic cultures. Mather and Babel (1959C) reported inhibition of slime formation caused by Pseudomonas fragi and Pseudo­monas putrefaciens when Leuconostoc cremoris was added to cottage cheese. In addition, the coliform organisms were inhibited. Elliker et al (1964) reported extension of shelf life of cottage cheese by the use of Streptococcus lactis subsp. diacetylactis which inhibited the spoilage bacteria.

Two antibiotics produced by Streptococcus lactis and Streptococcus cre­moris have been called nisin and diplococcin. Nisin has been isolated and found to possess variable inhibi­tory effects upon streptococci of groups A, B, E, F, G, K, M, and N, Micrococ­cus lysodeikticus, pneumococci, neisseria, some Bacillus, Clostridium, My­cobacterium, Lactobacillus, Actinomyces, Erysipelothrix species, and cer­tain staphylococci.

The lactobacilli have been demonstrated to possess potent antimicrobial properties against a wide variety of spoilage and pathogenic organisms. Inhibition of the growth of Staphylococcus aureus, Pseudomonas aeruginosa, Sarcina lutea, and Escherichia coli by lactobacilli has been observed in vitro Sabine (1963), Tramer (1966), DeKlerk and Coetzee (1961), and Mikolajcik and Hamdan (1975) demonstrated an antibiotic effect of Lactobacillus acidophi­lus culture on a wide variety of microorganisms.

Bryan (1965) reported inhibition of intestinal pathogenic and anaerobic putrefactive organisms by L. acidophilus and Z.bulgaricus. Inhibitory factors elaborated by lacto­bacilli have been termed lactolin, lactobrevin, lactocidin, lactobacillin, acidolin, and acidophilin.

It is not yet clear whether these compounds are individually antimetabolites or are related to each other. The various microbial antimetabolites elaborated by the cultures appear to contribute to the total preservative potential and spectrum displayed by fermented dairy products.