Introduction
Dairy starter cultures can be defined as lactic acid bacteria that are added to food intentionally to promote fermentation. Their key function is to stimulate the generation of lactic acid from a disaccharide known as lactose. The resultant acid produces an acidic pH that is responsible for the preservation of fermented milk or cheese (Gilliland 2018). The purpose of this paper is to describe dairy starter cultures and their use in cheese ripening. Information regarding their history, taxonomy, genetics, metabolism, types, production and commercial production are also explained.
Background Information on Dairy Starter Cultures
Dairy starter cultures result in the production of specific desirable properties in milk and its products by their normal growth process as well as the production of lactic acid (O’Malley 2016). Lactic acid produces a low pH that aids in the coagulation of milk protein. The general name for cultures that produce lactic acid is lactic acid bacteria. However, other starter microorganisms can be added for the purpose of flavor enhancement through the production of flavor chemicals like diacetyl. Starter microorganisms also contribute to the development of desired textures in fermented dairy products. Alterations in texture are attributed to the pH effect as well as the disintegration of fats, proteins, and other components of milk influence flavor and texture of cultured and/or aged products through the breakdown of proteins, fats, and other milk constituents (Celik and Tarakci, 2017). The low pH of fermented milk products serves to inhibit the growth of food spoilage microorganisms, thereby increasing the shelf life of the food commodities.
History of Dairy Starter Cultures
For a long time, fermented foods and beverages have been produced without using any industrial starter cultures. Conventional approaches to production entailed backslapping, which is a practice of introducing a small fraction of a previous batch of fermented food to initiate a new fermentation. Microorganisms that are inherent to specific food items were also exploited in the fermentation process without the need to introduce bacteria from external sources. Another approach that was commonly used was carrying out fermentation in special containers that permitted the preservation of starter culture microbes within crevices and pores. These methods made it possible to develop specific types of fermented items and are still applied currently in small-scale production facilities, home production, and less developed countries. However, the main shortcomings of these techniques were high chances of failure, slow fermentation, and contamination. Additionally, it was impossible to produce consistent results at all times because of fluctuations in the fermentation conditions and microbial populations. Contemporary large-scale industrial making of fermented commodities requires constant quality, strict quality control to guarantee food safety, and predictable manufacturing schedules.
Louis Pasteur discovered pure culture microbiological techniques in the 1860s, which served as the starting point for the development of microbial starter cultures (King-thom and Jong-kang 2017). In 1873, Joseph Lister came up with a pure culture of lactic acid bacteria that he referred to as Bacterium lactis (which was later known as Lactococcus lactis). From this point, research endeavors focused on the production of bacteria that were involved in the fermentation of dairy products., the function of pure cultures in the maturing of cream was ascertained in the 1880s following the work of three scientists named Storch, Weigman, and Conn in Denmark, Germany, and the USA, respectively. Thereafter, the role of flavor-enhancing bacteria in fermentation was established. Christian Hansen started a microbial culture business in 1878. This enterprise has progressed over the years and continues to supply starter cultures for various food applications such as brewing, meat, baking, wine, and dairy production.
The earliest commercial starter cultures were prepared by growing pure bacterial strains in milk that had undergone heat sterilization. The optimum pH for the growth of the microorganisms was maintained through the addition of calcium carbonate. However, the key shortcoming of these cultures was limited shelf lives because of a reduction in cell viability and fermentative potential. These problems led to the development of unrefined dry culture formulations. Further advancements involved the development of freeze-dried cultures. Nonetheless, this version of cultures needed to be grown in intermediate cultures. This setback motivated the development of frozen cultures that are in use at present. Technological improvements have also augmented freezing and drying methods to come up with improved starter cultures. The contemporary starter culture business has developed various microbes that can be applied to different food production processes compared to the initial cultures that were meant for the processing of milk commodities only.
Taxonomy of Dairy Starter Cultures
Dairy starter cultures have been renamed over time. However, they are still classified under two broad categories of mesophilic and thermophilic cultures. The first taxon is Streptococcus lactis that is now referred to as Lactococcus lactis sub-sp. lactic. The second type is Streptococcus cremoris that is currently known as Lactococcus lactis sub-sp. cremoris. Their major function is the production of acid during the manufacture of buttermilk, sour cream, and cheese. The third type is Streptococcus diacetylactis, which is now known as Lactococcus lactis sub-sp. lactis biovar. diacetylactis. Its function is acid and flavor production. The bacterium is commonly used in the production of ripened butter, sour cream, buttermilk, and cheese. Leuconostoc cremoris is currently called Leuconostoc mesenteroides sub-sp. cremoris, whereas the naming of Leuconostoc lactis remained unchanged. These two bacteria contribute to flavor and are commonly applied to the making of buttermilk, cottage cheese, sour cream, and ripened butter.
Mesophilic cultures have four species. The first two are Streptococcus thermophilus and Lactobacillus Helvetica whose names have not changed. Lactobacillus bulgaricus has changed to Lactobacillus delbrueckii sub-sp. bulgaricus, whereas Lactobacillus lactis is now called Lactobacillus delbrueckii sub-sp. lactic. These bacteria are used in the production of fermented milk, yogurts, Emmental, and Italian cheese.
Genetics of Starter Cultures
Extensive studies have been done over the last three decades to understand the genetics of starter bacteria. They include investigations on plasmid biology and molecular biology techniques, which have led to the explication of the entire bacterial genome. These discoveries have paved the way for metabolic manipulation of commercially important bacteria to enhance desirable traits and suppress unwanted ones (Frantzen and others 2018). One crucial trait that has been introduced to starter cultures is increased resistance to bacteriophage attacks, which is a desirable industrial feature.
The Lactococcus lactis subspecies lactis IL1403 genome has been sequenced and found to consist of a 2365-kb circular chromosome. It has a G+C content of 35.4% and possesses 2310 open reading frames (Felis and others 2017). This information has provided an insight into the genetics of lactococcal starters. When compared to other bacterial genomes such as Bacillus subtilis the Lactococcus lactis genome is significantly smaller, which suggests the specialized adaptation of Lactococcus to thrive in the nutrient-rich milk milieu.
Metabolism of Starter Cultures
The key metabolic pathway for starter cultures in the dairy industry is lactic acid fermentation of lactose, which is a biological pathway that involves the conversion of glucose, other hexoses, and disaccharides such as lactose and sucrose into energy in the form of adenosine diphosphate (ATP) and lactic acid. This process takes place in the absence of oxygen under the influence of the enzyme lactate dehydrogenase, which catalyzes the formation of lactate (lactic acid) from pyruvate (Gaenzle 2015). This reaction is reversible and is facilitated by NADH as electron carriers. Two main types of lactic acid fermentation are possible: homolactic and heterotactic. One molecule of glucose yields two molecules of lactate in homolactic fermentation, whereas the heterotactic process generates lactic acid in addition to ethanol and carbon dioxide gas through the phosphoketolase pathway.
Types of Starter Cultures
Starter cultures are grouped into two main classes: mesophilic starters and thermophilic starters. The first type demonstrates optimum growth at temperatures ranging from 21.1 to 32.2oC, whereas thermophilic cultures grow best between 37.7 and 46.1oC. These bacteria are further grouped into four types that are O-type, D-type, L-type, and LD-type. The O-type cultures have Lactococcus lactis subsp. lactis and Lactococcus lactis subsp. cremoris as the predominant bacteria. They are homofermentative because they only produce lactic acid. D-type has the O-type bacteria in addition to bacteria responsible for flavor such as Streptococcus lactic subsp. lactis var. diacetylactis. This flavor-enhancing bacterium leads to the production of cultured cream butter or buttermilk. Carbon dioxide is also produced and contributes to a delicate flavor in cheese. The L-type starter culture consists of O-type bacteria and Leuconostoc mesenteroides subsp. mesenteroides that impart flavor. The main flavor compounds generated by this culture are acetic acid, diacetyl, acetaldehyde, and carbon dioxide among others. However, the level of carbon dioxide produced by this type of culture is less than that generated by D-type. The LD-type is a blend of L-type and D-type bacteria to produce a unique and delicate aroma and flavor (Gilliland 2018).
Growth Inhibitors of Lactic Acid Bacteria
Even though lactic acid bacteria play a vital role in the industrial processing of food, fermentation cannot be allowed to proceed indefinitely because the continued growth of bacteria can cause food spoilage. Certain compounds have been reported to interfere with the growth of lactic acid bacteria and impair their fermentative capacity. They include fumaric acid, sorbic acid, nisin, decanoic acid, carbon dioxide, and sulfur dioxide (Rossi and Veneri 2016). Sulfur dioxide hampers the growth of lactic acid bacteria in cultures at a pH of 3.5 or below. Nisin, which is an antibiotic produced by the bacterium Streptococcus lactis, has been shown to inhibit a number of lactic acid bacteria such as lactobacilli, leuconostoc, and pneumococci. The effect of high levels of carbon dioxide on lactic acid bacteria is unknown. However, it is suggested that when carbon dioxide is in excess, fumarate does not undergo malolactic fermentation. Sorbic acid also demonstrates inhibitory upshots against lactic acid bacteria. It is suggested that inhibitors are potentiated by the addition of ethanol at concentrations of 8 to 10 %. However, the effectiveness of these inhibitors reduces as the size of the inoculum increases.
Bacteriophage
Bacteriophages (also referred to as phage) are microscopic viruses that are capable of attacking and destroying bacteria (Penadés and others 2015). Phages cannot reproduce without a host cell. The first step of phage infection is the attachment to the cell wall of the bacteria, leading to the insertion of phage DNA into the cell. The foreign phage DNA directs the host cell to produce phage parts such as capsids and coats. These parts are assembled to form new phage particles that then leave the bacterial cell after causing lysis and death. The rate of phage reproduction in host cells is so high that one bacterial cell can produce up to 200 bacteriophages that can infect other host cells. One notable characteristic regarding phages is that they are strain-specific, meaning that they can only attack specific bacterial strains. For these reasons, the rotation of cultures and resistance can be exploited as a means of controlling infection (Oliveira and others 2017).
Phage is pervasive and can access dairy factories through contaminated raw milk or carrier culture strains. Phage infection can lead to a phenomenon where no lactic acid is produced despite introducing starter cultures into a fermenting batch (‘dead vats’). Other causes of phage contamination are unsanitary cultures or residual cultures. Therefore, strict plant sanitation regimens and culture handling protocols should be observed to circumvent phage problems. Chemical disinfectants can be used to rid the milk of contaminating phage. Another alternative is heat treatments at temperatures ranging from 63 to 88°C for 30 minutes.
Commercial Production of Dairy Starter Cultures
Maintaining the quality of starter cultures is important because it can help to guarantee the consistency of the resultant product. Commercially produced cultures are mainly available in freeze-dried, spray-dried, or frozen versions. Starter cultures can be propagated in several ways in readiness for large-scale production. The first step involves preparing the commercial culture by growing in buffered media to promote maximum growth without compromising the production of acid. The ensuing cells are then concentrated by centrifugation after which they are lyophilized or stored at -40oC without significant reduction inactivity. However, such starter cultures have a long lag time compared to other types.
Production of commercial cultures often occurs in three states involving different cultures. The mother culture is the initial inoculation that produces other subsequent cultures. The intermediate culture is often used when producing larger quantities of starter cultures, whereas cultures in the bulk starter culture stage are the ones that are finally used in the production of dairy products.
To produce pure cultures, all preparations should be done in a separate room maintained at positive air pressure with the aid of 0.2 µm filters. All surfaces should be made of materials that can withstand sterilization. 200 ppm chlorine should be used for the sterilization of surfaces. Furthermore, any transfers need to be done using sterile pipettes. The most commonly used culture medium is pasteurized milk. However, three alternatives can be used, including reconstituted skimmed milk powder at concentrations of 10 to 12%. The milk should be tested and confirmed to be free of antibiotics. A second alternative is a mixture of whey and whey powder. Nonetheless, this mixture does not yield very good results compared to skim milk due to diminished buffer capacity. A third substitute is commercially produced culture media made from powdered milk proteins. High-quality milk free of antibiotics, large quantities of contaminating bacteria should be used. Rancid and milk from cows suffering from mastitis should be avoided. The culture medium should undergo heating for 60 minutes at approximately 88oC to get rid of bacteria and other contaminants. Heating also eliminates inhibitory substances and lowers the redox potential to promote the growth of lactic acid bacteria.
Phosphate buffers can be added to the medium to increase cell counts. Another advantage of phosphate is that it binds calcium ions that are essential or phage attachment to bacterial cells thus conferring resistance against attack by bacteriophage. This step should be omitted or avoided when dealing with Lactobacillus bulgaricus because the growth of this species is usually inhibited by the presence of phosphate ions. Using skim milk with low calcium levels or adding anhydrous ammonia also helps to impede phage.
At the end of the fermentation process, the pH should range from 4.5 to 5. Lower pH values encourage the bacteria to reach the stationary phase of growth, thus rendering them ineffective. After ascertaining that the correct pH and cell counts have been attained, the milk needs to be cooled to a temperature of 4oC. The final storage temperature is determined by the type of culture. For example, storage temperatures for thermophilic starters should not be lower than 20oC.
The Role of Cultures in Cheese Ripening
Lactic acid bacteria can be found naturally in milk for cheese making together with other microorganisms. Environmental contamination of unrefrigerated milk can introduce more bacteria. Therefore, it is possible to make cheese without inoculating it with additional cultures. However, the normal procedure during cheese making involves adding domestic cultures, which facilitates cheese production from pasteurized milk. In this context, culture denotes blends of bacteria, molds, and yeast that are introduced to cheese or milk. Cultures serve two crucial purposes in cheese making: development of acidity and promotion of ripening (Rossi and Veneri 2016). Lactic acid cultures fulfill both roles. However, several special cultures may be added to promote ripening.
As milk is fermented, lactic acid bacteria lead to the production of lactate, which alters the pH by lowering it. Acidification is important for the development of the characteristic cheese flavor and texture. It also enhances safety by preservation. Other functions of lactic acid include coagulation of milk proteins, promotion of syneresis, and preventing the growth of pathogenic and food spoilage bacteria. Acidic conditions induce and hasten the rate of coagulation. Syneresis refers to the regulation of moisture levels in cheese. Low pH leads to the contraction of the protein matrix in curd thereby expelling moisture. When it comes to the development of flavor, texture, and color, high pH (low acidity) gives rise to pliable, soapy, zesty, and bitter-tasting cheese, whereas increased acidity produces brittle cheese with blotchy color (Bekele and others 2019).
Cultures also promote the curing of cheese by producing growth factors that are essential to the development of non-starter microbes that play a role in the overall flavor. Lactic cultures secrete enzymes such as proteases and lipases that promote ripening in the inner parts of cheese. These enzymes also influence the texture and flavor. Special or secondary cultures may be needed for additional features such as surface ripening and development of ‘eyes.’
Conclusion
Starter cultures are collections of microorganisms that play a vital role in the industrial production of cheese and other dairy products. Their key mode of action is through the production of lactic acid during fermentation. In cheese production, cultures promote acidification, which enhances the flavor, texture, promotes ripening, and preserves the final product by preventing the growth of pathogenic and food spoilage microbes.
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