MD1396Z - Process for preserving the Saccharomyces cerevisiae CNMN-Y-21 yeast strain - Google Patents
Process for preserving the Saccharomyces cerevisiae CNMN-Y-21 yeast strain Download PDFInfo
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- MD1396Z MD1396Z MDS20180111A MDS20180111A MD1396Z MD 1396 Z MD1396 Z MD 1396Z MD S20180111 A MDS20180111 A MD S20180111A MD S20180111 A MDS20180111 A MD S20180111A MD 1396 Z MD1396 Z MD 1396Z
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- saccharomyces cerevisiae
- temperature
- yeast strain
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- 240000004808 Saccharomyces cerevisiae Species 0.000 title claims abstract description 33
- 235000014680 Saccharomyces cerevisiae Nutrition 0.000 title claims abstract description 31
- 238000000034 method Methods 0.000 title claims abstract description 14
- 239000002028 Biomass Substances 0.000 claims abstract description 21
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims abstract description 15
- 238000003860 storage Methods 0.000 claims abstract description 10
- 240000002900 Arthrospira platensis Species 0.000 claims abstract description 9
- 235000016425 Arthrospira platensis Nutrition 0.000 claims abstract description 9
- 235000020183 skimmed milk Nutrition 0.000 claims abstract description 9
- 239000000725 suspension Substances 0.000 claims abstract description 6
- 238000000605 extraction Methods 0.000 claims abstract description 4
- 238000004108 freeze drying Methods 0.000 claims abstract description 4
- 230000001476 alcoholic effect Effects 0.000 claims abstract description 3
- 238000007710 freezing Methods 0.000 claims abstract description 3
- 230000008014 freezing Effects 0.000 claims abstract description 3
- 235000015097 nutrients Nutrition 0.000 claims abstract description 3
- 239000000243 solution Substances 0.000 claims description 12
- 238000005119 centrifugation Methods 0.000 claims description 4
- 239000013049 sediment Substances 0.000 claims description 4
- 238000000926 separation method Methods 0.000 claims description 4
- 239000000126 substance Substances 0.000 claims description 4
- 241001464430 Cyanobacterium Species 0.000 claims description 3
- 238000000502 dialysis Methods 0.000 claims description 3
- 238000010790 dilution Methods 0.000 claims description 3
- 239000012895 dilution Substances 0.000 claims description 3
- 239000006228 supernatant Substances 0.000 claims description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 3
- 238000013019 agitation Methods 0.000 claims description 2
- 238000001035 drying Methods 0.000 claims description 2
- 150000001720 carbohydrates Chemical class 0.000 abstract description 17
- 102000004169 proteins and genes Human genes 0.000 abstract description 14
- 108090000623 proteins and genes Proteins 0.000 abstract description 14
- 229940082787 spirulina Drugs 0.000 abstract description 6
- 239000013543 active substance Substances 0.000 abstract description 2
- 230000007774 longterm Effects 0.000 abstract description 2
- 244000005700 microbiome Species 0.000 abstract description 2
- 235000014633 carbohydrates Nutrition 0.000 description 16
- 230000001681 protective effect Effects 0.000 description 7
- 238000004321 preservation Methods 0.000 description 6
- GXCLVBGFBYZDAG-UHFFFAOYSA-N N-[2-(1H-indol-3-yl)ethyl]-N-methylprop-2-en-1-amine Chemical compound CN(CCC1=CNC2=C1C=CC=C2)CC=C GXCLVBGFBYZDAG-UHFFFAOYSA-N 0.000 description 4
- 230000035790 physiological processes and functions Effects 0.000 description 3
- 230000000975 bioactive effect Effects 0.000 description 2
- 239000002244 precipitate Substances 0.000 description 2
- 238000003756 stirring Methods 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- 230000002225 anti-radical effect Effects 0.000 description 1
- 239000003963 antioxidant agent Substances 0.000 description 1
- 230000003078 antioxidant effect Effects 0.000 description 1
- 230000001143 conditioned effect Effects 0.000 description 1
- 238000009776 industrial production Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 230000000638 stimulation Effects 0.000 description 1
- 230000001502 supplementing effect Effects 0.000 description 1
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
Description
Invenţia se referă la biotehnologie, şi anume la un procedeu de conservare a tulpinii de levuri Saccharomyces cerevisiae CNMN-Y-21, care poate fi utilizat pentru păstrarea microorganismelor pe un termen îndelungat şi utilizarea acestora ca surse de substanţe biologic active. The invention relates to biotechnology, namely to a process for preserving the yeast strain Saccharomyces cerevisiae CNMN-Y-21, which can be used for long-term preservation of microorganisms and their use as sources of biologically active substances.
În prezent este cunoscut un procedeu de conservare a tulpinii de levuri Saccharomyces cerevisiae CNMN-Y-21 pe mediul protector constituit din lapte degresat, care după un an de păstrare în stare liofilizată, după 3 pasaje de cultivare, când tulpina revine la starea sa fiziologică normală, asigură obţinerea unor valori ale conţinutului de proteine şi carbohidraţi de 35,32% şi 23,08% din biomasa absolut uscată, respectiv [1]. Currently, a process is known for preserving the yeast strain Saccharomyces cerevisiae CNMN-Y-21 on a protective medium consisting of skimmed milk, which after one year of storage in a lyophilized state, after 3 cultivation passages, when the strain returns to its normal physiological state, ensures obtaining protein and carbohydrate content values of 35.32% and 23.08% of the absolutely dry biomass, respectively [1].
Dezavantajul acestui procedeu constă în aceea că conţinutul substanţelor bioactive (proteinelor şi carbohidraţilor) după un an de conservare în stare liofilizată, este insuficient de înalt. The disadvantage of this process is that the content of bioactive substances (proteins and carbohydrates) after a year of preservation in a lyophilized state is insufficiently high.
Problema pe care o rezolvă prezenta invenţie constă în elaborarea unui procedeu de conservare a tulpinii de levuri Saccharomyces cerevisiae CNMN-Y-21 prin liofilizarea acesteia, cu utilizarea unui mediu protector ce asigură stimularea conţinutului de proteine şi carbohidraţi după un an de conservare. The problem solved by the present invention consists in developing a process for preserving the yeast strain Saccharomyces cerevisiae CNMN-Y-21 by lyophilizing it, using a protective medium that ensures the stimulation of protein and carbohydrate content after one year of preservation.
Procedeul revendicat constă în cultivarea tulpinii de levuri Saccharomyces cerevisiae CNMN-Y-21 pe un mediu nutritiv în decurs de 72 de ore la temperatura de 26-28°C, suspensionarea culturii până la titrul de 105-106 ml-1 într-un mediu constituit din lapte degresat şi 5-10% vol. soluţie care conţine 10 mg/ml de extract, obţinut din biomasa cianobacteriei Spirulina platensis CNMN-CB-02, congelarea rapidă a suspensiei la temperatura de -20°C, liofilizarea şi păstrarea la temperatura de 4°C, totodată extractul este obţinut prin extragerea biomasei cu soluţie alcoolică de 20-30% cu separarea sedimentului, uscarea la temperatura 40-450C, extragerea repetată cu soluţie de NaOH de 0,45% timp de 60 şi 30 min la agitare cu centrifugarea, separarea sedimentelor şi unirea supernatantelor, dializa până la pH 7,5-8,5 şi diluarea cu apă până la concentraţia de 1% mas. de substanţe uscate. The claimed process consists of cultivating the yeast strain Saccharomyces cerevisiae CNMN-Y-21 on a nutrient medium for 72 hours at a temperature of 26-28°C, suspending the culture to a titer of 105-106 ml-1 in a medium consisting of skimmed milk and 5-10% vol. solution containing 10 mg/ml of extract, obtained from the biomass of the cyanobacterium Spirulina platensis CNMN-CB-02, rapid freezing of the suspension at a temperature of -20°C, lyophilization and storage at a temperature of 4°C, at the same time the extract is obtained by extracting the biomass with a 20-30% alcoholic solution with separation of the sediment, drying at a temperature of 40-45°C, repeated extraction with a 0.45% NaOH solution for 60 and 30 min under agitation with centrifugation, separation of the sediment and union of the supernatants, dialysis to pH 7.5-8.5 and dilution with water to a concentration of 1% mass of dry substances.
După un an de conservare în stare liofilizată conţinutul de proteine atinge valori de 47,79-48,40% din BAU iar cel de carbohidraţi de 31,30-32,21% din BAU. After one year of preservation in a lyophilized state, the protein content reaches values of 47.79-48.40% of BAU and the carbohydrate content reaches 31.30-32.21% of BAU.
Suplinirea mediului protector cu extract bioactiv obţinut din biomasa cianobacteriei Spirulina platensis ce posedă acţiune antioxidantă şi antiradicalică pronunţată a contribuit la majorarea conţinutului de proteine şi carbohidraţi în biomasa de levuri Saccharomyces cerevisiae CNMN-Y-21. Supplementing the protective medium with a bioactive extract obtained from the biomass of the cyanobacterium Spirulina platensis, which possesses pronounced antioxidant and antiradical action, contributed to increasing the protein and carbohydrate content in the biomass of yeast Saccharomyces cerevisiae CNMN-Y-21.
Rezultatul tehnic al invenţiei constă în majorarea conţinutului de proteine şi carbohidraţi în biomasa de levuri Saccharomyces cerevisiae CNMN-Y-21 după un an de păstrare în stare liofilizată respectiv cu 35,31-37,03% şi 35,62-39,56%. The technical result of the invention consists in increasing the protein and carbohydrate content in the biomass of Saccharomyces cerevisiae CNMN-Y-21 yeasts after one year of storage in a lyophilized state by 35.31-37.03% and 35.62-39.56%, respectively.
Rezultatul tehnic al invenţiei este condiţionat de faptul că pentru prima dată în calitate de mediu protector pentru liofilizarea levurilor Saccharomyces cerevisiae CNMN-Y-21 se utilizează laptele degresat combinat cu extractul biologic activ obţinut din biomasa de spirulină. The technical result of the invention is conditioned by the fact that for the first time, skimmed milk combined with the biologically active extract obtained from spirulina biomass is used as a protective medium for lyophilization of Saccharomyces cerevisiae CNMN-Y-21 yeast.
Exemple de realizare a invenţiei Examples of embodiments of the invention
Exemplul 1 Example 1
Procedeul de obţinere a extractului din biomasa de spirulină se realizează în felul următor. La 1000 ml de biomasă de spirulină (100 mg/ml) se adaugă 1000 ml alcool de 30%, se agită timp de 60 min şi se centrifughează la 3500 rot/min timp de 20 min. Precipitatul obţinut este uscat la 40-50°C şi supus extracţiei cu 500 ml de soluţie de NaOH de 0,45%, timp de 60 min la agitare permanentă. După centrifugarea la 3500 rot/min la precipitat se adaugă 200 ml de soluţie de NaOH de 0,45% şi se extrage timp de 30 min. După centrifugare supernatantele se reunesc şi sunt supuse dializei până la pH 7,5-8,5 şi diluarea cu apă până la concentraţia de 1% mas. de substanţe uscate. Extractul obţinut este supus sterilizării. The process of obtaining the extract from spirulina biomass is carried out as follows. To 1000 ml of spirulina biomass (100 mg/ml) add 1000 ml of 30% alcohol, stir for 60 min and centrifuge at 3500 rpm for 20 min. The precipitate obtained is dried at 40-50°C and subjected to extraction with 500 ml of 0.45% NaOH solution, for 60 min with permanent stirring. After centrifugation at 3500 rpm, 200 ml of 0.45% NaOH solution is added to the precipitate and extracted for 30 min. After centrifugation, the supernatants are combined and subjected to dialysis to pH 7.5-8.5 and dilution with water to a concentration of 1% by mass of dry substances. The extract obtained is subjected to sterilization.
Exemplul 2 Example 2
În tuburi înclinate cu mediu malţ agarizat se cultivă tulpina Saccharomyces cerevisiae CNMN-Y-21, timp de 72 ore la t° de 26-28°C, după care cultura se suspensionează în mediul protector constituit din lapte degresat şi 5% vol. de extract obţinut din biomasa de spirulină, până la titrul de 105- 106 UFC ml-1. Suspensia obţinută se repartizează câte 1,0 ml în flacoane, se congelează rapid la -20°C, după care se liofilizează. Probele liofilizate sunt sigilate şi plasate în frigider la t° de 4°C. După un an de păstrare în stare liofilizată tulpina se cultivă pe mediul malţ agarizat timp de 3 pasaje consecutive, pentru a reveni la starea fiziologică normală şi se determină conţinutul de proteine şi carbohidraţi în biomasă. In inclined tubes with malt agarized medium, the Saccharomyces cerevisiae strain CNMN-Y-21 is cultivated for 72 hours at t° of 26-28°C, after which the culture is suspended in the protective medium consisting of skimmed milk and 5% vol. of extract obtained from spirulina biomass, up to a titer of 105-106 UFC ml-1. The suspension obtained is distributed in 1.0 ml vials, quickly frozen at -20°C, then lyophilized. The lyophilized samples are sealed and placed in a refrigerator at t° of 4°C. After one year of storage in a lyophilized state, the strain is cultivated on malt agarized medium for 3 consecutive passages, to return to the normal physiological state and the protein and carbohydrate content in the biomass is determined.
Biomasa obţinută conţine 48,40% proteine şi 32,21% carbohidraţi din BAU, faţă de 35,32% proteine şi 23,08% carbohidraţi, conform celei mai apropiate soluţii. The obtained biomass contains 48.40% protein and 32.21% carbohydrates from BAU, compared to 35.32% protein and 23.08% carbohydrates, according to the closest solution.
Exemplul 3 Example 3
În tuburi înclinate cu mediu malţ agarizat se cultivă tulpina Saccharomyces cerevisiae CNMN-Y-21, timp de 72 ore la t° de 26-280C, după care cultura se suspensionează în mediul protector constituit din lapte degresat şi 10% vol. extract obţinut din biomasa de spirulină, până la titrul de 105-106 UFC ml-1. Suspensia obţinută se repartizează câte 1,0 ml în flacoane, se congelează rapid la -200C, după care se liofilizează. Probele liofilizate sunt sigilate şi plasate în frigider la t0 de 40C. După un an de păstrare în stare liofilizată tulpina se cultivă pe mediul malţ agarizat timp de 3 pasaje consecutive, pentru a reveni la starea fiziologică normală şi se determină conţinutul de proteine şi carbohidraţi în biomasă. In inclined tubes with malt agarized medium, the Saccharomyces cerevisiae strain CNMN-Y-21 is cultivated for 72 hours at t° of 26-280C, after which the culture is suspended in the protective medium consisting of skimmed milk and 10% vol. extract obtained from spirulina biomass, up to a titer of 105-106 UFC ml-1. The suspension obtained is distributed in 1.0 ml vials, quickly frozen at -200C, then lyophilized. The lyophilized samples are sealed and placed in a refrigerator at t0 of 40C. After one year of storage in a lyophilized state, the strain is cultivated on malt agarized medium for 3 consecutive passages, to return to the normal physiological state and the protein and carbohydrate content in the biomass is determined.
Biomasa obţinută conţine 47,79% proteine şi 31,30% carbohidraţi din BAU, faţă de 35,32% proteine şi 23,08% carbohidraţi, conform celei mai apropiate soluţii. The obtained biomass contains 47.79% protein and 31.30% carbohydrates from BAU, compared to 35.32% protein and 23.08% carbohydrates, according to the closest solution.
Sporirea conţinutului de proteine şi carbohidraţi în biomasa de levuri Saccharomyces cerevisiae CNMN-Y-21 în rezultatul conservării pot pune în evidenţă proprietăţile tehnologice a acesteia pentru ulterioara utilizare la producerea industrială. The increase in protein and carbohydrate content in the biomass of Saccharomyces cerevisiae CNMN-Y-21 yeast as a result of preservation can highlight its technological properties for further use in industrial production.
Tabel Table
Conţinutul de proteine şi carbohidraţi în biomasa de levuri Saccharomyces cerevisiae CNMN-Y-21, după un an de conservare în stare liofilizată Protein and carbohydrate content in the biomass of yeast Saccharomyces cerevisiae CNMN-Y-21, after one year of preservation in lyophilized state
Procedeul utilizat Mediul protector Conc. Conţinutul de proteine Conţinutul de carbohidraţi % BAU % M % BAU % M Conform celei mai apropiate soluţii Lapte degresat - 35,32 100,00 23,08 100,00 Conform procedeului revendicat Lapte degresat + extract biologic activ 5% 48,40 137,03 32,21 139,56 10% 47,79 135,31 31,30 135,62Process used Protective medium Conc. Protein content Carbohydrate content % BAU % M % BAU % M According to the closest solution Skimmed milk - 35.32 100.00 23.08 100.00 According to the claimed process Skimmed milk + biologically active extract 5% 48.40 137.03 32.21 139.56 10% 47.79 135.31 31.30 135.62
Datele din tabel demonstrează majorarea cu 35,31-37,03% a conţinutului de proteine şi cu 35,62-39,56% a conţinutului de carbohidraţi în biomasa de levuri Saccharomyces cerevisiae CNMN-Y-21 în procedeul propus în invenţie faţă de procedeul descris în cea mai apropiată soluţie. The data in the table demonstrate the increase by 35.31-37.03% of the protein content and by 35.62-39.56% of the carbohydrate content in the biomass of Saccharomyces cerevisiae CNMN-Y-21 yeasts in the process proposed in the invention compared to the process described in the closest solution.
1. Батыр Л., Джур С., Бырса М., Рошка М. Изменение биохимического состава некоторых культур дрожжей после года хранения в лиофилизированном состоянии. XII Международная научно-практическая конференция European Scientific Conference. Пенза, 7.11.2018, p. 12-15 1. Batyr Л., Джур С., Бырса М., Рошка М. Changes in the biochemical composition of some yeast cultures after a year of storage in a lyophilized state. XII International scientific-practical conference European Scientific Conference. Penza, 7.11.2018, p. 12-15
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