Lactobacillus delbrueckii subsp. bulgaricus
Lactobacillus delbrueckii subsp. bulgaricus ( Gram stain)
Lactobacillus delbrueckii subsp. bulgaricus ( Lactobacillus delbrueckii subsp bulgaricus advertised as short form is also L. delbrueckii ssp. bulgaricus common) is a Gram-positive, rod-shaped bacterium that is used for the production of yogurt. Here, the bacterium exploits the existing in milk lactose ( milk sugar) into lactic acid fermentation, the lactic acid formed in this process leads to acidification and curdling the milk.
It is a subspecies ( subspecies ) of Lactobacillus delbrueckii. Previously, the bacterium under the name Lactobacillus bulgaricus was known. This term goes back to the Bulgarian doctor Stamen Grigorov, who discovered the bacterium in 1905. The subspecies includes numerous tribes. The genome of the strain Lactobacillus delbrueckii subsp. bulgaricus ATCC 11842 was completely sequenced in 2006.
- 3.1 Outer systematics
- 3.2 Internal systematics
- 3.3 Etymology
- 4.1 yogurt production
- 4.2 Other Dairy Products
- 5.1 Literature
- 5.2 Notes and references
Features
Appearance
L. delbrueckii subsp. bulgaricus is a Gram-positive medium- long, rod-shaped bacterium. A single cell is 2.0 to 9.0 microns ( micrometers) long and 0.5 to 0.8 micron wide. In light microscopic image individual cells, but also short chains of cells are found in a row. The bacterium does not possess flagella for active movement and can not form Überdauerungsformen as endospores.
In pure culture it forms on solid media containing glucose - circular, colorless to appear opaque colonies that are small with a diameter of 2 to 5 mm.
Growth and metabolism
As a typical representative of the lactic acid bacteria lactobacilli grow anaerobically, but aerotolerant, that is, they grow in the presence of atmospheric oxygen, but do not require oxygen for their metabolism. They are catalase - negative and oxidase - negative. However, they are able to form cytochromes when cultured on nutrient media containing hemins or blood. In this case, they then exhibit a positive reaction in the oxidase test. Further, a typical lactic acid bacteria, the need for complex flag growth factors and amino acids in the culture.
L. delbrueckii subsp. bulgaricus can be used as a typical lactic acid bacteria in a fermentation utilize various carbohydrates for energy. This subspecies is able to utilize, glucose, fructose and lactose. Features of the fermentation ( fermentation ), in that the substrates are degraded without oxygen. Typical of lactic acid bacteria in the fermentation product is lactic acid, this pathway is therefore referred to as lactic acid fermentation. Since lactic acid or lactate ( the anion of lactic acid) is the main product from this fermentation, L. delbrueckii subsp is. bulgaricus counted among the homofermentative Lactobacillaceae.
It has aldolase, a key enzyme in the glycolytic pathway, therefore they can reduce glucose in the glycolysis. Other hexoses such as fructose are converted to glucose for it. The disaccharide is lactose (milk sugar) is cleaved with the aid of the bacterial enzyme β -galactosidase own in the two components of glucose and galactose. Galactose is converted to glucose by the enzyme UDP-glucose 4-epimerase. L. delbrueckii ssp. bulgaricus has this enzyme.
In glycolysis, one molecule of glucose is then broken down into two molecules of pyruvate. This is then reduced by the enzyme lactate dehydrogenase with the formed in the oxidation of coenzyme NADH to glyceraldehyde lactate, NADH is oxidized to NAD . Thus, two molecules of lactic acid are whole, from one molecule of glucose formed. Here, the energy gain is two molecules of ATP per molecule of glucose (see figure).
The subsp Lactobacillus delbrueckii. bulgaricus existing lactate is stereospecific, so that in this reaction, almost exclusively D-(- ) -lactate ( Syn: (R) -lactate ) produced. The proportion of D- (-)- lactic acid is 96.0 to 99.4 %. This enantiomer is known as left-turning lactic acid, in contrast to the dextrorotatory lactic acid, L- ( )- lactate ( Syn: (S) -lactate ). The mentioned sporadically in yogurt products statements " with dextrorotatory lactic acid" or " with dextrorotatory cultures " refer to this distinction.
In addition to the main product lactate you can still find small amounts ( in total less than 10%) of acetate, carbon dioxide, ethanol or acetoin. The acetaldehyde contained as a flavoring agent in yogurt in small amounts is not by L. delbrueckii ssp. bulgaricus formed, but by also present in starter cultures Streptococcus thermophilus.
Temperatures usually used for the cultivation in the range of 30-40 ° C, the bacteria still grow well at 45 ° C, the optimum temperature of an examined strain is 42 ° C. Thus, the bacterium is one of the mesophilic organisms, with a tendency to thermophilicity. The optimum pH for growth is ( 5.2 to 6.2 pH) with acidic pH values up to pH 4.0 can be tolerated at a slightly acidic pH. By the produced lactic acid, the pH of the culture medium decreases if no buffering additives are included.
Some enzymes used in the metabolism, in order to reduce certain substrates are detected in a "Bunte number " to identify a bacterium. Here Lactobacillus delbrueckii subsp records. bulgaricus is characterized in that it does not have many of these enzymes. He has neither catalase nor oxidase. Nitrate reduction through the enzyme nitrate reductase (NADH ) (EC 1.7.1.1 ) is not possible, nor is the degradation of proteins ( proteins ) by proteolytic enzymes, or the degradation of urea by the enzyme urease. The enzyme β -galactosidase is present and allows him the growth in milk under utilization of the lactose contained therein.
Genetics
The genome of numerous strains of the bacterium has been completely sequenced. The first sequencing was carried out in 2006 of Lactobacillus delbrueckii subsp. bulgaricus ATCC 11842, which applies to the subspecies as a representative strain and can be attributed to a bacterial strain that was used in 1919 in Bulgarian yogurt. The genome has a size of 1865 kilobase pairs (kb ), which is only 40 % of the genome size of Escherichia coli. There are 1529 proteins annotated. The small genome size is a further indication of their adaptation to the habitat milk. There are many complex growth factors such as amino acids and vitamins are present, that the bacteria had lost the ability to synthesize a number of metabolites over time.
In 2011, the study of the genome of the strain L. delbrueckii ssp occurred. bulgaricus 2038, which has long been used in industrial yogurt production. The differences with the previously examined strains of the subspecies as well as other members of the genus Lactobacillus have been considered in more detail. Compared with the first strain examined the genome size of 1873 kb is slightly larger. Lactobacillus delbrueckii subsp. bulgaricus 2038 is, for example, in contrast to the other strains capable of synthesizing the amino acid lysine.
The results of sequencing show a GC content ( the fraction of nucleic bases guanine and cytosine) in the bacterial DNA of about 50 mole percent, which is significantly more than in other Lactobacillus species, such as L. acidophilus, the GC content of DNA is 34 to 37 mole percent.
Pathogenicity
L. delbrueckii (including subspecies ) is not pathogenic, it has long been used in the food industry. It is assigned to risk group 1 By Biostoffverordnung in connection with the TRBA 466.
Evidence
The bacterium is well in MRS broth cultivated, which is incubated microaerophilic to anaerobic at a temperature of 37 ° C. A selective medium is not available, however, a selective enrichment may be accomplished when the medium (up to pH 4.0) has a low pH and contains carbohydrates, to a tomato juice -peptone agar is used, for example. For detecting lactic acid bacteria China Blue Lactose Agar (CLA) is often used, as well, the bacteria colonies are stained as a result of lactic acid formation blue.
Biochemical tests for the identification and detection of enzymes in the metabolism can be used, in addition, microscopic examination type information. An accurate identification to the species level or subspecies is not guaranteed in this way.
Occurrence
The natural habitat of L. delbrueckii ssp. bulgaricus is the milk and products made from them. Fermented milk products have long been used because they are more durable than fresh milk. 1905 isolated the Bulgarian microbiologist and physician Stamen Grigorov in yogurt a previously unknown bacterium, which he later " Bacillus bulgaricus " and called on the current nomenclature L. delbrueckii ssp. bulgaricus is. In dairies, it is used as a starter culture in the production of yoghurt ( Streptococcus salivarius in combination with subsp. Thermophilus ), as well as in the production of cottage cheese and sour milk cheese.
System
Outer systematics
Inside systematics
The nomenclature of the bacterium has changed several times in the 20th century. The original name " Bacillus bulgaricus " is based on the discovery by the Bulgarian microbiologist and physician Grigorow. 1919, the bacterium by Orla Jensen and was described as Thermobacterium bulgaricum. As a result of the investigations of Rogosa and Hansen at different Lactobacillus species from the year 1971, the name was changed in Lactobacillus bulgaricus. In 1984, was the change in the allocation to the species L. delbrueckii as a subspecies of Lactobacillus delbrueckii subsp. bulgaricus ( Orla -Jensen 1919) and White et al. 1984 comb. nov.
Etymology
The genus name is now used can be attributed to the presence and appearance of the bacterial cells, lac from the Latin meaning milk while bacillus (Latin ) refers to the rod- like shape. The species name is a tribute to Max Delbrück, during the term of the subspecies on the origin of the bacteria concerns of Bulgarian yogurt.
Industrial importance
Yoghurt production
L. delbrueckii ssp. bulgaricus is used in the production of traditional yoghurt. In dairies it is usually in combination with Streptococcus salivarius subsp. thermophilus (Streptococcus thermophilus ) specific starter culture is added to pasteurized and homogenized milk. The mixture is incubated at 43-45 ° C, by the lactic acid fermentation occurs within a few hours acidification and curdling the milk. The lactic acid production of the starter culture, the pH in the final yogurt is lowered to pH 4.0 to 4.2, and the food containing the fermentation after about 0.7-1.1 % lactic acid.
This classic yogurt is characterized by a sour taste. One other Lactobacillus species, V.A. L. acidophilus produced sour milk product is characterized by a milder taste. It was sold under the names " acidophilus milk " or " Bioghurt " increasingly popular with the German consumer. According to the Regulation on milk products, this product is referred to as " mild yogurt ", by the altered starter culture containing this product mainly dextrorotatory lactic acid, ie L-( )- lactic acid ( Syn: ( S)- lactic acid). This enantiomer is the more suitable for the human physiological form, as in human metabolism of L-lactic acid is formed, and this is broken down by a specific enzyme, L-( ) -lactate dehydrogenase, faster. The traditional yogurt with Lactobacillus delbrueckii subsp. bulgaricus is however still widespread in southern European countries.
Other dairy products
Lactobacillus delbrueckii ssp. bulgaricus is also used in the production of cottage cheese and sour milk cheese. The beginning of the process is similar as in the production of yoghurt. The protein casein contained in milk is thereby precipitated - it clots, this is achieved in the production of sour milk cheese using lactic acid bacteria. In the thermophilic starter cultures Streptococcus salivarius subsp. thermophilus used in combination with L. delbrueckii ssp. bulgaricus or L. delbrueckii subspecies ssp. lactis, in individual cases, other Lactobacillus species. The mixture is incubated at 32-45 ° C.
A drink used in Asian cuisine is Kumys ( alternate spelling: Kumiss ). Also this made from mare's milk fermented milk containing L. delbrueckii ssp. bulgaricus, together with certain yeasts that perform alcoholic fermentation, thus contains the beverage in addition to lactic acid, ethanol and carbon dioxide.