Iran, the potential source of dairy starter microorganisms

Content

Preface

1. Foundation and Backgrounds
1.1 Introduction
1.2 History
1.3 Dairy starter cultures
1.4 The reaction between dairy yeasts and LAB
1.5 Ecology of dairy starter bacteria
1.5.1 Lactic Starter cultures
1.5.1.1 Mesophilic starter cultures
1.5.1.2 Thermophilic starter cultures
1.5.2 Non-lactic starter cultures
1.6 Classification of starter microorganism
1.6.1 Dairy starter bacteria
1.6.1.1 Lactobacillus genus
1.6.1.2 Streptococcus genus
1.6.1.3 Leuconostoc genus
1.6.1.4 Lactococcus genus
1.6.1.5 Enterococcus genus
1.6.1.6 Brevibacterium genus
1.6.1.7 Pediococcus genus
1.6.2 Dairy starter yeasts
1.6.2.1 Saccharomyces genus
1.6.2.2 Candida genus
1.6.2.3 Schizosaccharomyces genus
1.6.2.4 Kluyveromyces genus
1.6.3 Dairy starter molds
1.6.3.1 Penicillium genus
1.6.3.2 Geotrichum genus
1.7 Starter cultures function
1.8 Grow starter bacteria in milk
1.9 Treating starter cultures in dairy products
1.10 Bacteriophages in the dairy industries
1.10.1 Control the number of phages
1.11 Factors affecting the performance of starter cultures
1.11.1 Internal factors
1.11.2 External factors
1.11.3 Subsidiary factors

2. Materials and methods
2.1 Appliance
2.2 Mediums
2.3 Antibiotics
2.4 Chemical solutions and organic materials
2.5 Carbohydrates
2.6 Dairy samples
2.7 Collection and coding of samples
2.8 Preparation, enrichment and isolation of the strains
2.9 Carbohydrate fermentation and hydrolysis of L-arginine, starch, casein and nitrate reduction
2.10 Investigating antibiotic resistance
2.11 Assessment of proteolytic and lipolytic activity
2.12 Evaluation of acid production

3. Results and discussion
3.1 Isolation and identification of strains
3.2 Antibiotic susceptibility of strains
3.3 Investigating proteolytic and lipolytic activities
3.4 Investigating acidification activity

4. Conclusion

Suggestions

References

Figures

Figure 1. Carbohydrate fermentation test

Figure 2. Antibiogram test

Figure 3. Acid production test

Figure 4. Biochemical characteristics of Lactobacillus, Bacillus and Saccharomyces strains

Figure 5. The pH changes at time intervals between 0 and 24 h in acidification test for Lactobacillus and Bacillus and Saccharomyces strains

Figure 6. The pH changes in acidification test (a): Lc. lactis; (b): Lc. plantarum; (c): Lc. garvieae

Tables

Table 1. Strain Coding Guide

Table 2. Biochemical characteristics of Lactobacillus, Bacillus and Saccharomyces strains

Table 3. Biochemical characteristics of Lactococcus strains

Table 4 . Carbohydrates fermentation test of Lactobacillus, Bacillus and Saccharomyces strains

Table 5. Carbohydrates fermentation test of Lactococcus strains

Table 6. Resistance and sensitivity of Lactobacillus and Bacillus strains to different antibiotics

Table 7. Resistance and sensitivity of Lactococcus strains toward different antibiotics

Table 8. Proteolytic and lipolytic activities of Lactobacillus and Bacillus and Saccharomyces strains.

Table 9. Proteolytic and lipolytic activities of Lactococcus strains 60

Acknowledgment

Thanks to my god, who gave me the blessing of learning science, and helped me to deal with difficulties in order to finish this thesis successfully. During the course of my studies and in the preparation of this thesis, I have benefited from the guidance and assistance of the dear professors and friends that here, I need to thank all of them from deep of my heart and wish health and success for them. Dr. Mrs. Parvaneh Jafari, as one of the best professors in the field of Microbiology, Dr. Ahmad Ali Pourbabaei, who advise me as a consultant, and Dr. Zolfaghari, director of the Department of Microbiology and all of the ones helped me in this way.

With best wishes

Farzad Rahmati

Dedicated to

My dear parents, who, with their endless supports, provided me with the possibility of my development, and with their sacrifice taught me the concept of faith, love and how to live. I hope to be able to compensate for their indescribable affection.

Preface

Today, the consumption of milk and its products in each community is one of the most important development indicators. Based on the latest scientific findings in the study of the nutritional value of milk and its products, in particular fermented products, the annual consumption of 200 liters of this valuable foodstuff result in longer life, more physical and intellectual efficacy, reduction of infectious diseases, reduction of bone diseases and desirable growth of children and teens. Of course, the full realization of these features depends on the consumption of milk throughout their lives. The results of the research conducted by the Ministry of Health and Medical Education of Iran indicate that the Iranian people from 2 to 3 units of daily intake, only use 0.7 units of milk and its products, while in other countries, especially in Europe, this is not the case. Currently, in countries where milk and its products are consumed in large quantities, much of the calcium is provided. For example, in the United States and Canada, three-quarters of the calcium diet is supplied through milk and its products. The importance of dairy products is becoming more prominent when it comes to the fact that in Iran, calcium is the second the most common substance after iron, which is not observed in their regime and milk consumption in our country is lower than the WHO^[1] and FAO^[2] recommendations. Undoubtedly, one of the reasons for the low level of dairy consumption in Iran is the lack of a sufficient variety of these products and the lack of compatibility with the taste of the people. Recalling the many health-therapeutic properties of milk is not just a good reason to encourage many people, especially children, and adolescents, to consume dairy products. For this purpose, we have to produce flavored products in line with the taste of the Iranian people, encouraging people to consume more dairy products. With increasing population growth and richer culture of dairy consumption related factories produce diverse dairy products which relied on import starter cultures from abroad. Contrary to what was said, not much research has not been done yet in Iran about dairy. Studies conducted in the field of the LAB^[3] are more focused on the dairy industry to optimize biochemical attributes, quality of dairy products and identification.

The aims of the present research are isolation, identification, and study of technological attributes of Lactococcus, Lactobacillus, Bacillus and Saccharomyces Cerevisiae starter strains from traditional dairy products in the Lorestan province, a region of Iran. By this study not only, we are able to preserve these worthwhile strains for extensive commercial exploitation and prevent the annual withdrawal of millions of dollars from the country, but also produce dairy products according to the taste of the Iranian.

1. Foundation and Backgrounds

1.1 Introduction

Milk and its products are valuable sources of water (87%), proteins (3.5%), fat (3.5%), carbohydrates (5% lactose) vitamins A،D،K،E, B1،B2،B6،B12, elements such as calcium, phosphorus and energy [1]. Milk is a white liquid which produced from the mammalian. It`s the only source of mammalian foods of birth, and can even be a valuable source of food for adults, especially the elderly, and also plays an important role in nutrition due to calcium and protein. This product is the most valuable food ingredient and has almost all the ingredients necessary for human growth and survival; it is the only food that can balance more humanly nutritional needs and is considered as one of the most important sources of animal proteins. It contains a variety of valuable vitamins and minerals, most notably calcium, which plays an essential role in the formation and strengthening of the bones, the health of teeth, hair and reducing cardiovascular diseases and is the enemy of cancer and type II diabetes [2]. Proteins are one of the most important components of milk and play an important role in the production of milk products. The nutritional, physiological and functional effects of proteins are one of the things that can be mentioned. Among the functional properties of proteins, including thermal stability for products requiring a thermal process, gel formation, especially for products such as cheese and fermented products, rheological properties, surface activity, and water absorption. Some bacteria that are cultivated in milk and convert lactose to lactic acid, which changes the flavor and milk texture. By converting lactose to lactic acid, the pH of the milk is reduced, thus preventing the growth and survival of pathogenic bacteria. On the other hand, reducing the amount of lactose allows the use of these products for people with lactose intolerance. In the fermentation industry, lactose is decomposed by the starter and new fermentation products such as yogurt and cheese are produced. Raw milk is a good environment for many microorganisms due to its sufficient moisture, near-neutral pH, and richness of the food. Milk has a very small number of bacteria when taken from a healthy cow breast, or in other words, the microbial burden is less than 1000 bacteria per ml. Of course, this value varies greatly in different livestock, and in some cases even up to 15,000 microorganisms per ml. The products obtained from fermented milk have different flavors and, due to less water than milk, they are more resistant to food corruption. Fermented milk is produced in appropriate temperature conditions, and depending on the type of yeast or bacteria, milk type, and process conditions, different products are produced [3]. Many antimicrobial compounds like lactoperoxidase and agglutinin are present in melted butter, but they are rapidly inhibited by fermentation of sugar and acid production by common bacteria. Therefore, many microorganisms are capable of growing in such an environment, although some of these microorganisms, such as some species of lactic LAB, like Lactococcus and Lactobacillus are useful for the production of various milk products [3].

1.2 History

The history of dairy starter use is very long. Some old books also refer to the use of fermented dairy products in the BCE, which suggests that the appearance and use of dairy starter has a long history. Farmers who lived in England about six thousand years ago, like modern humans, kept cows and sheep and other livestock because of their economic value. A chemical analysis of the pottery remains found in fourteen ancient regions in the United Kingdom reinforced this long-standing cognitive theory that primary farmers kept from cows and sheep not only because of their meat but also because of their dairy products. In the latest research conducted by the chemist Richard Evershed from the University of Bristol, the recent mass spectrometry technique was used to identify the remaining milk fats in the old pottery. Although in Ancient India and Persia, we referred to yogurt in 500 BC, and in Iranian culture, people have believed that Abraham, the Prophet, has had a long life with health due to our regular and daily consumption. But the first of yogurts might have been accidentally prepared, when the milk was sliced into the bags of goat's skin, the bacteria in the goat's skin, the fermented milk, and the yogurt was obtained. A Russian biologist believed that Lactobacillus was good for health, and he tried to promote the use of yogurt throughout Europe. Following this, in 1919, the first industrial production factory for yogurt was built in the city of Barcelona, Spain, and yogurt consumption increased day by day.

As the population and the culture of dairy products grows, we have more than thousands of dairy products currently in dairy industries. Due to the increasing consumption of dairy products, including yogurt and cheese, the need for LAB as starter cultures is also increased. The field and statistical surveys of dairy factories have shown that the industries in Iran are completely dependent on the supply of these bacteria from abroad, according to the latest annual data of two million dollars for importing starter cultures from to the country [4]. This dependency is such that, if there is a limitation on the sale of starter to Iran, dairy factories will not be able to produce dairy products. On the other hand, with the development of the dairy industry in the country, along with the promotion of the health culture of foods and the probability of contamination in traditional dairy products, the consumption of industrial dairy products in all parts of the country has replaced the consumption of traditional dairy products, so the traditional production of the above products is dramatically being destroyed. Examining different dimensions of this problem shows that the elimination of these strains can be considered as an irreparable loss of nationality and even global.

1.3 Dairy starter cultures

Starter cultures are the microorganisms that are added to dairy products to create fundamental changes in all the properties, such as aroma, flavor, and product texture [5]. As noted earlier, dairy starter cultures use lactose to produce lactic acid and by acidifying the milk and lowering the pH, breaks down the milk protein, casein. This is due to the collapse of protein particles after reaching the isoelectric point. Changes that occur produce flavoring substances Includes diacetyl, which, in addition to creating flavors, creates a special taste of cream that makes the product very tasty. Today, various factories are engaged in the production of industrial starter cultures, which, by sending to the dairy industry, produce different types of dairy fermented products. In general, the use of microorganisms can reduce the aw^[4] change the pH, produce inhibitor compounds like alcohol and bacteriocins eliminate the foodstuff that is easily consumed by corrosive organisms. Of course, today, in terms of food fermentation, process conditions, and maintenance, they create an environment in which certain types of microorganisms grow and, instead of corruption, leave a positive effect on the quality of the foodstuff. Most fermented food is produced by the activity of LAB and fungi, especially bacteria, yeasts and, to a lesser extent, molds. All three of these groups have similar ecological characteristics that are capable of growing in low pH and aw, although only bacteria of lactic acid and optional yeasts can grow and multiply under anaerobic conditions, as a result of which these microorganisms are often found in fermented products while they work together. The order of decomposition of matter among foods seems to be the following: first, carbohydrates, then proteins, and after which the fats are attacked by microorganisms, and among the carbohydrates, the sugars, then the alcohols and finally the acids are used. The reactions in which protein is decomposed into proteins is referred to as proteolytic or disintegrating, and the reactions in which fatty matter is decomposed is called lipolytic [6]. Fermentative microorganisms, carbohydrates and their derivatives to a degree Highly converted to alcohols, acids, and carbon dioxide. In addition, fermentation processes have long-term stability and also create flavor, aroma and optimum texture. In addition, when they are produced sufficiently, the alcohols and acids derived from fermentation from growth and the effect of proteolytic and lipolytic microorganisms that can corrode food. In addition, because of competition in the consumption of existing foods, they also affect the proliferation. However, if the acids produced by fermentation against oxygen, they can break down by mildew and lose their inhibitory effect on microorganisms. To give Characteristics of the fermentation microorganisms are that they have the potential to produce many enzymes that control the chemical reactions during fermentation. In fact, 1 g of the microorganism (based on dry weight), which has a high enzyme production capacity, can break down 10000 g lactose per hour [7]. In lactic fermentation, bacteria produce lactic acid using the food in the environment. By producing this acid, conditions are favorable for the growth of susceptible microorganisms and thereby prevent their growth. However, the amount of lactic acid produced in this type of fermentation is not so much that it can alone take on the role of food protection. The role of preservative lactic fermentation is in due to the production of several materials with their protective properties and their synergistic effect in this regard [7].

As already mentioned, starter cultures produce lactic acid, which has a very important effect on the quality of the product in terms of texture, taste, moisture content, and the absence of pathogenic microbes and toxins. The amount of lactic acid production by these microbial strains is very important in the production of some products, such as Cheddar cheese. Important characteristics of starter cultures can be a high rate of growth and dominance over the environment in a way that does not allow the growth of microorganisms to cause corrosion. Starter cultures should have a large number of beneficial microorganisms, as well as maintain their activity throughout all stages of fermentation [8]. In dairy factories, the medium used for the starter is a non-fat dry milk. Reclaimed non-fat milk and add water to it, then sterilize it and use it. Low-fat dry milk is used to grow Leuconostoc and Streptococcus.

Today, fermentation is widely used to produce various food products among vegetables, dairy products such as cheese, yogurt, and meat.

1.4 The reaction between dairy yeasts and LAB

During the production and delivery of fermented dairy products, yeasts grow well in low aw and high salt concentrations due to their low pH tolerance. Such growth can have negative effects and contribute to corruption and even allergic reactions and consumer toxicity. At the same time, the growth of yeasts necessarily improves the texture and aroma of fermented dairy products, which brings about the proteolytic and lipolytic activity. In fact, yeasts are essential for the production of kefir, Kumis and various types of Camembert cheeses. Finally, sulfur compounds are produced by yeast in the cheese. For any effect of fermented dairy products, yeasts need to reach a high density and must compete with other existing microorganisms, but mainly in lactic acid bacteria, positive and negative reactions have been reported among yeast and lactic acid bacteria, Though their mechanism is well understood. The positive reaction between them is the production of carbon dioxide, pyruvate, propionate, and succinate.

Types of lactic acid bacteria have nutritional requirements in many compounds and also stimulate the synthesis of vitamins by producing amino acids and yeast. In addition, some lactic acid bacteria release galactose that can facilitate the growth of negative lactose yeasts [9]. Negative reactions are mainly caused by leaky inhibitory growth. Yeasts prevent the production of bacterial lactic acid compounds, such as phenylalanine, 4-hydroxy phenylalanine and silicic acid peptides. Lactic acid production of bacteria is inhibited by the production of fatty acids, and it is essential to metabolize lipolytic yeasts, the effects of positive and negative reactions on the growth and metabolism of lactic acid bacteria or yeasts, and the development of growth or perfume production.

1.5 Ecology of dairy starter bacteria

Most of the starter plants used today are from the family of lactic bacteria that form part of the flora of milk in its natural state. These bacteria are likely to be of plant origin, such as Lactococcus, or like Bifidobacterium and Lactobacillus acidophilus, which has an intestinal origin. Today's starter cultures are the result of keeping some milk or cream from batch production on the previous day, and inoculation as a starter for the next day's production. This procedure is called different names, but the term " Back-Slopping " is the most well-known term used in this method. Generally, starter cultures are divided into two groups [8]. I. Lactic starter cultures

II. Non-lactic starter cultures

1.5.1 Lactic Starter cultures

Lactic starter cultures are generally bacterial cultures and are divided into two groups:

1.5.1.1 Mesophilic starter cultures

Their appropriate growth temperature is 30-30° C, including Lactococcus Lactis subsp. Cremoris, Lactococcus Lactis subsp .Lacti s, Leuconostoc Mesenteroides subsp .Cremoris, Leuconostoc Lactis, and Streptococcus Diacetilactis. Mesophilic starter cultures have the following categorizations: 1. The combination of strains used in it: ii) Combination of uncertain and indeterminate strains. ii) Specific single, double or multiple strains. 2. In terms of taste and aroma production characteristics: i) Type L or B, only a flavoring producer.

ii) Type D, merely the taste producer. iii) Type LD or BD, merely flavor producer. iv) Type O, without flavor, and merely acid producer.

1.5.1.2 Thermophilic starter cultures

Thermophilic starter cultures with a suitable growth temperature of 40-45 °C are the most important ones, including Streptococcus Thermophlilus, Lactobacillus Delbrueckii subsp. Bulgaricus, Lactobacillus Helveticus and Lactobacillus Delbrueckii subsp .Lactis [8].

1.5.2 Non-lactic starter cultures

Unlike the first group, they are moldy and used to produce cheese. The method is to use them in combination with the raw milk or to place it on the cheese. This group is responsible for the production of proteolysis and cheese ripping includes Penicillium Camemberti, Penicillium Candidum, Geotrichum Candidum in the production of white mildew cheeses such as Camembert and Barry, Penicillium Roquefort in the production of blue streak cheeses such as Roquefort and Stilton [8].

1.6 Classification of starter microorganism

1.6.1 Dairy starter bacteria

With the advent of molecular biology, genetic methods are increasingly being used to study the genetic similarities between different bacterial groups. These techniques have led to significant changes in the classification of the LAB. The LAB are divided into two genera as previously mentioned. Bacterial starter cultures are categorized into eight different genus includes Enterococcus, Lactococcus, Lactobacillus, Leuconostoc, Bifidobacterium, Corynebacterium, Pediococcus and Streptococcus. Of course, there are doubts as to whether the Enterococcus group is in the category of starter cultures, but since they are present in some industrial starter cultures, they are therefore classified in this category. Concerns about Enterococcus are due to the pathogenicity of a group of them [6]. Of course, they are able to exchange resistance genes to antibiotics (especially glycopeptide antibiotics such as vancomycin), and this raises further concerns about them. Vancomycin is one of the rarest antibiotics that act on bacteria that are resistant to several antibiotics. Bifidobacterium is not a family of the LAB. Although Bifidobacterium is sometimes placed in this family due to the production of lactic acid, so they are also explained.

1.6.1.1 Lactobacillus genus

This genus is a large group of gram-positive bacteria, negative catalase, and bacilli. Some species are homofermentative, while others are heterofermentative. While some species produce L-type of glucose, others produce D-type of lactate. Some species are also racemic, which means, produce production, both L, and D types. Occasionally, some of its species are morphological, cocci and there are errors in their diagnosis with Leuconostoc and even Lactococcus. Lactobacillus are starter culture used in the production of yogurt and cheese. They are also used as a starter to improve and accelerate the ripping of Cheddar and similar cheeses, to reduce bitterness. L . delbrueckii subsp . bulgaricus is widely used in conjunction with Streptococcus thermophilus as the starter culture of yogurt. This subtype is homofermentative and produces 2% (w/v) of lactic acid. The optimum growth temperature is 42 °C, also grows at 45° C and above. They have not grown in low salt concentrations and are susceptible to bile salts. L. acidophilus, naturally occurring in the intestine, is also commonly used as a starter, and of course, this is more of a probiotic bacterium. It is homofermentative and produces high levels of lactic acid of type D in milk. Its optimum growth temperature is 37 °C and is relatively capable of bearing oxygen (in comparison to the Bifidobacteria, which is often used in conjunction with the bacteria in the products). At below 20 °C, they have less growth and most strains do not grow at a temperature of 15 °C. Since this bacterium produces D-type lactic acid, there are concerns about its use in infant food [10]. Lactobacillus Casei is one of the natural habitats of the small intestine and is resistant to bile and is used as a probiotic which, of course, is present in a series of starter cultures and is commonly known as one of the non-starter lactic bacteria found in Cheddar cheese. L-type Lactate is the major isomer produced by this bacterium. However, some strains of this bacterium produce a small amount of D-type lactate because of weak racemic activity. L. helveticus Sometimes, along with some other thermophilic LAB in some fermented dairy products such as Effin cheese, Mozzarella and yogurt are used. One of the advantages of using this bacterium, along with L . delbrueckii subsp . bulgaricus is that the strain is capable of consuming galactose. Therefore, in products that need to be sugar-free, the use of the bacterium is useful [10]. Since most of the strain has a quasi-proline amino-peptidase activity, this bacterium is used in the production of modified cheddar cheeses, with the creation of characteristics such as the sweetness of Swiss cheeses like Emmental. Recently, certain strains of this bacterium have been used as a starter to reduce bitterness or to improve the flavor in a group of cheeses or to speed up the process. These bacterial species are homofermentative and produce high levels of lactic acid D/L in the milk. Most of its strain grows at 45 °C, and most strains do not have any growth or very slight growth at a temperature of 15 °C. Some of the specific strain may last for about a few weeks at a temperature of 15 °C or less [11]. L . delbrueckii subsp. lactis, L . delbrueckii subsp . bulgaricus and homofermentative thermophiles in combination with Streptococcus Salivarius subsp .Thermophilus, is used as the starter culture for Swiss cheeses, Parmesan and Mozzarella. Beta-galactosidase is the dominant enzyme in L. helveticus, L . delbrueckii subsp. lactis and L . delbrueckii subsp . bulgaricus. In mixed cultures, compared with single cultures, coccobacilli produce more acid and improve the taste. The free amino acids released from casein with the help of proteases from L . delbrueckii subsp. bulgaricus stimulate Streptococcus thermophilus growth. In contrast, Str. thermophilus produces carbon dioxide and formate that stimulates the growth of L . delbrueckii subsp. bulgaricus. At the beginning of the incubation, Str. thermophilus grows faster and separates the excess oxygen and produces the mentioned stimulants, then Str. thermophilus growth is reduced because of the concentration of lactic acid increases and the amount of L . delbrueckii subsp. bulgaricus that can withstand more acid is increased. A number of lactobacilli have been isolated from cheese, the most famous of which are L. casei, Lactobacillus Brevis, Lactobacillus Fermentum, and these latter bacteria are heterofermentative and can cause defects of Cheddar cheese. Adding homofermentative Lactobacillus accelerates the process of ripping the cheese [11].

1.6.1.2 Streptococcus genus

This genus is diplococcus, short chains or long chains, according to the species and conditions of growth., all of them are homofermentative, produce acid without gas. According to Lancefield's theory, they are classified according to the antigenic characteristics of groups A to V. According to another Sherman's theory, these bacteria are divided into four groups includes Pyogenes, Viridians, Enterococcus, Lactis [6]. In lactis group, there are many varieties used as starter cultures in the production of dairy products, including Streptococcus Cremoris and Streptococcus lactis. The growth of these bacteria stops at 45° C, with the salt concentration of 4.5% and a significant contribution to the production of flavor [12]. Str. thermophilus, a thermophilic bacterium grows at 45 °C and above, is widely used in the production of yogurt, Mozzarella cheese, and some other cheeses. Since the mid-1990s, it has been used to produce Cheddar cheese. This bacterium, along with lactic acid, is used in some DVS / DVI starter systems and produces acid at the heating stage and may, of course, be used as a double step to protect the bacteriophage. Like Lactococcus and most Leuconostoc and Str. thermophilus strains are negative catalase, cocci and in pairs or chains, and most of them generally create long chains. This bacterium produces L-type lactic acid and is not able to produce Co2 from the fermentation of glucose. Some strains of these bacteria produce urease and also can produce carbon dioxide from it. Since Str. thermophilus can grow in pasteurizers, many of them are found in cheese. Urease-producing strains have the ability to open and crack cheese. Strains of this bacterium differ in their ability to use galactose [8]. The use of strains that are not capable of fermenting galactose causes large quantities of these reducing sugars to remain in the product. Since this sugar and other reducing sugars are involved with amine acids in the Millard reaction, it is, therefore advisable to use galactose-capable strains to reduce the likelihood of color changes caused by the Millard reaction in the heated dairy product. Streptococcus, found in saliva, is called Str. salivarius, which is very similar to Str. thermophilus. For this reason, Str. thermophilus was known as Str. salivarius for many years. Today, it is clear that despite the high similarity between these two species, there are enough differences that can be categorized into two different species. Str. thermophilus is sensitive to low salt levels and also in an environment of high osmotic power.

1.6.1.3 euconostoc genus

This genus in a sucrose-containing environment, create a slippery capsule of dextran, which is problematic due to blockage of pipes and drains. Leuconostoc is heterofermentative and produces many aromatic compounds, especially Diacetyl. The Co2 gas, which is one of the metabolites produced by this bacterium, is produced in large quantities, causing unpleasant cavities and protuberance in the cheese. Species of this genus include Leuconostoc Dextranicum, Leuconostoc Citreum and Leuconostoc Mesenteroides, which have the properties of osmophilic and grow in concentrations of 60% sucrose as well as high salt concentrations. The first two species are used to prepare butter and cheese as a starter. Based on microbiological studies, Leuconostoc is generally Gram-positive, cocci, and in terms of shape and size (short or double chains), they are similar to Lactococcus. Unlike Lactococcus, Leuconostoc does not produce ammonia from arginine and produce only D-type lactic acid. With some exceptions, Leuconostoc has a poor growth in milk and can`t reduce litmus prior to coagulation in the litmus milk medium [6]. Leuconostoc are important flavoring manufacturers of dairy fermentation products. Two important starter species of this genus are the L. citreum and L. mesenteroides and Leuconostoc Lactis. Unlike Lactococcus, Leuconostoc is heterofermentative and produce Co2 from glucose and fructose. While Co2 production in Cheddar is undesirable, gas production in some other products, such as Emmental cheese, is desirable. It is difficult to isolate and identify Leuconostoc in the starter cultures. Carbohydrate fermentation and identification of acid lactic isomers are important and useful in identifying them. Leuconostoc do not change much in pure cultures in milk and are generally considered to be ineffective. Although Leuconostoc ferments the dairy products, they do it so slowly. Lactobacillus with Leuconostoc has a synergistic effect on the production of diacetyl from the citrate found in the milk [13]. Lactococcus, which quickly fermented lactose in milk, facilitate to absorb citrate by the Leuconostoc. These bacteria have the necessary enzymes for fermentation of citrate to diacetyl, but acid-producing Lactococcus do not have these enzymes. Thus, the production of diacetyl by mesophilic lactic acid starter indicates the synergic activity of Lactococcus and Leuconostoc. In choosing Leuconostoc species in composite starter cultures, functional compatibility with Lactococcus should first be determined. Otherwise, the starter will fail to create the taste of the product. Low temperatures are necessary for the growth of both bacteria so that the growth of both Leuconostoc and Lactococcus has been observed at these temperatures. The natural habitat of Leuconostoc, like Lactococcus, is vegetables that contain fermentable sugars. Morphologically, Leuconostoc is circular cells that lie in long chains. These bacteria exhibit abnormal resistance to relatively high concentrations of vancomycin antibiotics in the range of about 500 mg/ml.

1.6.1.4 Lactococcus genus

Generally, the bacteria of this group were previously classified as Streptococcus species. They differentiated from other Streptococcus, and this distinction was due to their specific reaction to the N anti-serum group, the heat and salt tolerance and their color [8]. Lactococcus is a relatively new grouping of classification principles. Five species were stratified from the S treptococcus genus to make the Lactococcus genus. Only Lactococcus Lactis is used in dairy fermentation. Recent categorization relies on the apparent, biochemical, and molecular characteristics of living organisms. Lancefield grouped the bacteria in the older genus, Streptococcus, based on their cell wall carbohydrate serology. The Sherman Lactic Group bacteria are in the N Lansfield Group. When Lactococcus was classified into Streptococcus, they have undergone several changes. Recently, a distinctive physiological characteristic has been reported between the subtypes of Lc. lactis and Lc. cremoris. Strains belonging to Lc. lactis subtypes are capable of decarboxylating glutamate [13]. Lactococcus are spherical cells morphologically and appear in short chains, but they can also be found in the form of a pair of distinct cells. Some species, especially those that are vulnerable to the agglutinins found in the milk, demonstrate long chains. Lactococcus are Gram-positive and microaerophilic, have no catalase and are fermented [14].

1.6.1.5 Enterococcus genus

The most important genus Enterococcus is Streptococcus Faecalis. It is a Gram-positive species and is a negative catalase that tends to form chains of varying lengths. Their natural habitat is in the human gastrointestinal tract and other animals, and are considered as an indicator of fecal contamination in microbiology. Some species of this genus are pathogenic and apart from their ability to grow at 45 °C, pH 9.9 and their presence at high salt concentrations are similar to those of Lactococcus [8].

1.6.1.6 Brevibacterium genus

Gram-positive bacteria that are labial, cocci and occasionally cortical, aerobic, micro-aortic to anaerobic. The Brevibacterium Linens are the only one that has been studied in the dairy industry. The bacteria of this genus are Gram-positive, aerobic to facultative anaerobic and non-motility. It is important species are Brevibacterium Erythrogenes and B. linens which are used in cheese making for orange-red pigmentation at cheese levels such as Limburger. These bacteria have a relatively high proteolysis and amino acid breakdown and play a very important role in the process of ripping and flavoring in cheeses such as Camembert that their surface can be washed [6]. Determining the role of these bacteria in proteolysis of the final stage of ripping cheese is difficult. B. Linens decompose non-proteolytic casein, in particular, casein β. The pH is appropriate for this process of 7.4 and its effect on the final stages of the cheese-making process [15].

1.6.1.7 Pediococcus genus

The members of this genus are single, double, or four cocci, homofermentative, which have grown well in the 5.5% salt concentration, but slowly grow in 10% concentration and the their optimal growth temperature is 25-32 °C. Salt tolerant, acid production and a wide range of growth temperatures, especially at low temperatures, can be applied it to fermented food industries especially in the production of Canadian cheddar and other cheeses. An interesting feature of Pediococcus is an antimicrobial activity of some strains against Listeria Monocytogenes, Staphylococcus Aureus, and Clostridium Perfringens [16].

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^[1] 1. World Health Organization

^[2] 2. Food and Agriculture Organization

^[3] 3. Lactic Acid Bacteria

^[4] 4. Activity of the Water