Оптимізація процесів культивування у виробництві пробіотичних препаратів на основі лактобацил

Установлено, что степень лизируемости клеток значительно увеличивается при лимитировании углеводного субстрата и 0,5% сахарозы повышает литическую чувствительность клеток в 1,5 раза. Рост на средах, содержащих продукты гидролиза соевой муки и казеина, обработанных смесью протеолитических ферментов повышал литическую чувствительность клеток в 2 раза.

Определено, что комплексные среды обеспечивают образование культурами всего спектра незаменимых аминокислот. Определено, что культуральные жидкости обоих штаммов обладают высоким уровнем противоопухолевого действия, которое в 1,5 раза усиливается при использовании казеиновых ферментолизатов. Установлено, что среды с ферментолизатами казеина обеспечивают образование клеточной массы с выраженным иммуностимулирующим действием.

Установлено, что восприимчивость к действию литических ферментов усиливается в присутствии твина-80 и ионов йода в 1,5-2 раза, а антагонистическая активность - в 4-8 раз. Полученные нами результаты, позволили разработать технологические рекомендаии для включения в нормативно - техническую документацию на производства биомассы L. delbrueckii subsp. bulgaricus 51 и L. delbrueckii subsp. bulgaricus 86.

Ключевые слова: молочнокислые бактерии, пробиотики, ростовые факторы, лизоцим, литическая восприимчивость, культивирование, антагонистическая активность, противоопухолевое действие, иммуномодулирующее действие.

Abstract

Аzizpour kh. “The Оptimizаtion of processes cultivation for probiotics preparation production on the bases of lactic acid bacteria”. - Manuscript.

The dissertation for a Candidate's degree in Biological Sciences on specialty of 03.00.20- biotechnology.-Institute of Cell Biology and Genetic Engineering of National Academy of Sciences of Ukraine, Kyiv, 2004.

The dissertation presents the results of experimental researches for the optimization of processes in cultivation of lactic acid bacteria for probiotics preparation production.

As result of conducted experiments, executed by us, have allowed to establish series of leading technological parameters. which are interdependent with accumulation of biomass, formation of cell biotherapeutic potential, and sensitivity of cell walls of culture to operating lytic ferment complexes.

The experiments, which we conducted allowed us to restore a biological potency of an initial producer and to receive new version of the culture, which surpassed a stock strain on series of parameters (L. delbrueckii subsp.bulgaricus 86)

Thus, having extended outcomes of conducted researches, we determine the optimum culture conditions for L. delbrueckii subsp. bulgaricus 51. The conclusions have been made, that process of cultivation under production conditions are expedient for conducting are at 39±1^oС, with initial рН values of 6.5-7.0, and introducing a young inoculum in concentration not less than 10 % from volume medium fermentation. The cultivation should be conducted without forced aeration, but with constant stirring of fluid cultural in a regimen of 40 r.p.m, space factor fermenter 0.75, and usage of the multi-layered blender.

The analysis outcome of conducted researches have the referenced conditions for the definition of degree lysis of cells L. delbrueckii subsp. bulgaricus 51 by lysozyme are selected has shown, concentration of a lysozyme in a reaction mixture -100 мкг/ml, temperature of hydrolysis +25⁰С, duration - 1 hour, and also fixed more heat of hydrolysis, about 35 ч45⁰С, large duration of process of hydrolysis, about 3 - 5 hours it is to use, in practical work for realization of enzymatic hydrolysis of biomass L. delbrueckii subsp. bulgaricus 51 by lysozyme with the purpose of obtaining biologically fissile peptidoglycan.

Analysis of the influencing carbonic sources, nitrogenous feeding and different growth factors have allowed us to establish that by the most acceptable energy source for L. delbrueckii subsp. bulgaricus 51 appear to be glucose at 1 %. This can be obtained as result of an enzymatic or lead-acid hydrolysis of series of carbohydrate substratums - saccharose and starch. The most metabolized sources of nitrogen for the strain are the organic matters. Maximum productivity of the biomass demonstrated on the mediums keeping soybean extracts, peptone, hydrolysates of dry milk, cystine in a quantity of 0.1%, and yeast extract. The body height of the culture increased, as did also, its attachment on the medium of biotin and ions Fe²⁺.

At the same time, the development of L. delbrueckii subsp. bulgaricus 51 was inhibited in the presence of corn extract, inorganic sources of nitrogen, and also ions Co²⁺, and Zn²⁺.

We established that the degree of lysis cells increase considerably in limited amounts of carbohydrate substratum, and concentrations of saccharose (0.5%) in the medium provides decrease in degree of lysis cells by 1,5 times greater, than at other quantities of carbohydrate substratum.

At the same time, the decrease in degree of lytic sensibility was marked when the follow were present peptone in the medium, dry milk, corn and yeast extracts, cystine, ions Fe²⁺ and Zn²⁺. The evaluation of sensibility of this cell culture obtained from different culture conditions has shown. That the most stable to operating lysozyme of the producer cell is formed on medium MRS.

Twice more the sensitivity to operating lysozyme was exhibited by cells. Which evolve on complex hydrolysates keeping hydrolysates of soybean flour and casein, treated by admixture ferment preparations.

As it was possible to establish, the cultivar fluid of the strain L. delbrueckii subsp.bulgaricus 51, obtained, as result of the development of the producer on complex fermentation mediums, kept, in fair quantities all spectrum of irreplaceable amino acids.

The dependence of formation of carriers of antitumoral activity in cells lactobacillus from structure of medium culture and, first of all, naturally of organic source of nitrogenous feeding is rotined.

The versions of cells and their lysates obtained after cultivation on fermentation mediums, keeping only casein fermentolysate, and showed antitumoral activity at 2.4-4 times less than expressed.

It should be emphasised, that in cultivar fluid of both strains (L. delbrueckii subsp. bulgaricus 51 and L. delbrueckii subsp. bulgaricus 86) are at a sufficient level of antitumoral operating, which was saved in case of usage for casein fermentolysate, more than 1.5 times exceeded the capacity to growth inhibition of swelling both living cells, and their lysates.

It has appeared, that the mediums of cultivation in the structure which entered casein fermentolysate, provided formation of cell mass with expressed immunomodulators actions.

The change of dynamics during the accumulation of biomass and formation of biological properties under the effect of the twin-80 and potassium iodidum, wearing for all cultures personal nature, is rotined.

Fixed, that the sensibility of operating lytic enzymes strengthens in the presence of the twin-80 and ions of iodum at 1.5-2 times and antagonism activity - at 4-8 times.

Thus, the outcome, obtained by us, was that of complex research of the property series of lactobacillus cultures. This was conducted with the purpose of building an effective biotechnological process for obtaining, on their basis, biological preparations including probiotics. All have allowed to develop the technological recommendation for incorporation in technological documentation for production of the biomass L. delbrueckii subsp. bulgaricus 51 and L. delbrueckii subsp. bulgaricus 86.

Key words: lactic acid bacteria, probiotic, growth factors, lysozyme, cultivation, lytic sensibility, antagonism activities, antitumoral activity, immunomodulator action.

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