В результате проведенных исследований установлено, что активизированные культуры пропионовокислых бактерий обладают высокими антимутагенными и адгезивными свойствами, синтезируют значительное количество корриноидов и гемсодержащих ферментов. Выявлено, что с повышением концентрации железа в среде увеличивается синтез внеклеточных метаболитов, способствующих адаптации культур к металлу. Определены оптимальные технологические параметры выделения казеиновых фосфопептидов. Доказана высокая способность казеиновых фосфопептидов солюбилизировать двухвалентное железо. Установлена взаимосвязь между концентрацией железа и степенью солюбилизации. Отмечено, что железо, хелатированное казеиновыми фосфопептидами, сохраняется в двухвалентной форме в течение длительного срока хранения.
пропионовокислые бактерии, каталаза, пероксидаза, суперокиддисмутаза, казеиновые фосфопептиды, солюбилизация железа.
INTRODUCTION
The concept of optimal nutrition implies adequate organism supply with both macro- and micronutrients, including the essential microelements, particularly iron, as a key prerequisite for preservation of human health. Iron-deficient conditions remain a topical and untreated issue of modern medicine. Lack of iron in the organism leads to many negative consequences. One of them is the development of iron deficiency anemia [1].
Taking into account that man consumes iron in vegetable and animal products in everyday life and the presence of amino acids and peptides, as well as proteins of animal origin, promotes intake of the microelement, enrichment of diets with organic forms of iron seems reasonable. In our opinion, propionic acid bacteria, which possess the ability to synthesize considerable amounts of heme-containing enzymes and corrinoids thus increasing iron uptake, are the most convenient object for development of biotechnological production of iron in organic form [2].
Iron is known to be consumed only in the form of Fe2+. However, divalent iron undergoes chemical oxidation to an insoluble, nonassimilable trivalent form. To preserve bioavailability of iron, role of chelating agents, which promote solubilization of minerals preserving their soluble state, is of interest. Casein phosphopeptides (CPPs) are among the representatives of the chelators. CPPs are phosphorylated peptides formed from caseins of cow milk upon digestion by proteases [3, 4, 5]. Casein phosphopeptides are still poorly studied as both chelating agents and potential nutriceutics for human nutrition. Besides, there are no data in literature on the effect of CPPs on iron solubilization. Therefore, studies on iron-binding capacity of CPPs are of interest.
The aim of the work was to study the effect of various concentrations of iron sulfate on growth and biosynthesis of extracellular metabolites by propionic acid bacteria, as well as the study on chelating properties of casein phosphopeptides.
MATERIALS AND METHODS
Bacteria and culturing conditions. Cultures of the following propionic acid bacteria (PABs) strains were subject of the study: Propionibacterium freudenrichii subsp. shermanii AC-2503, Propionibacterium freudenrichii subsp. freudenrichii AC-2500, Propionibacterium cyclohexanicum Kusano AC-2260, and Propionibacterium cyclohexanicum Kusano AC-2259, all obtained from the All-Russian Collection of Microorganisms of the Institute of Biochemistry and Physiology of Microorganisms (Moscow) and activated by a unique biotechnology method developed in the East Siberian State University of Technology and Management. Divalent salt (FeSO4) was used as iron source. Propionic acid bacteria were cultured in serum medium supplemented with growth factors [6]. One-day culture grown on low-fat milk was used as an inoculate. Iron sulfate was added to the growth medium at concentration of 0.25--0.55 mg/mL. Propionic acid bacteria were cultured in the presence of iron sulfate for 24 h at 30°C. Culture growth kinetics was calculated according to a custom method.
Analytical procedures. The process of iron binding was followed by the amount of chelated Fe2+ (% iron remaining in divalent form to the total initial dose). Content of Fe2+ was determined using a reference method [7]. Content of Fe3+ was determined by spectrophotometry. The technique was developed according to the Industry-Specific Standard 34-70-953.4-88. The method is based on the interaction of dissolved iron with sulfosalicylic acid and measurement of optical density of the colored solutions thus formed.
Determination of extracellular metabolites was performed in the end of the exponential growth phase. Catalase activity was determined using a colorimetric technique [8], peroxidase activity, by spectrophotometry using the o-dianisidine reagent [9], and that of superoxide dismuatse, by autoxidation of adrenalin [10].
Antimutagenic activity was determined by the Ames test [2]; adhesion properties were studied on formalinized erythrocytes according to the in-depth Brilis technique; strain adhesiveness was estimated according to the index of microorganism adhesiveness (IMA) [11]; concentration of exopolysaccharides was estimated with anthrone reagent [12]; and vitamin B12 content was determined by spectrophotometry [13].
Solution of casein phosphopeptides was obtained by enzymatic hydrolysis of sodium caseinate. Metal-binding ability of CPPs depends on the extent of phosphorylation. To obtain hydrolysate with the maximal content of low-molecular weight phosphorylated peptides and free amino acids capable of formation of soluble complexes with iron, we redefined process parameters of CPP isolation. One-stage hydrolysis of Na caseinate with pepsin and trypsin with varying hydrolysis time was used. Molecular weight distribution of peptides in the aqueous solution of caseine phosphopeptides was evaluated by moderate pressure size exclusion chromatography on a TSK GEL (0.8/30 cm) column. Chelated iron content was determined by mass spectrometry. Tables discuss statistically significant differences at p < 0.05.
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