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БИОЛОГИЧЕСКИЕ НАУКИ
ISOLATION AND IDENTIFICATION OF YEASTS FROMAIRAG (KOUMISS) _AND KHOORMOG, TRADITIONAL DRINKS OF MONGOLIA_
Odontuya Bazarkhuu1, Nandin-Erdene Baasandorj2, Delgermaa Sovd3, Dulguun Dorjgotov4
'MSc student of Microbiology, 2Assistant lecturer,3Assistantprofessor,4Senior lecturer; Department of Biotechnology and Nutrition;
Mongolian University of Science and Technology
ABSTRACT:
Identification of yeasts by molecular biological methods was more reliable than a method based on carbon assimilation. In this study, we isolated and identified nine different yeast strains (Galactomyces geotrichum, Cryp-tococcus sp., Kluyveromyces marxianus, Kluyveromyces lactis, Rhodotorulla mucilaginosa, Candida zeylaoides and three Saccharomyces cerevisiae) from airag and khoormog.Two of them have P-galactosidase activity which means they can be used in the utilization of whey.
Key words: Yeast ITS sequence, API 20C AUX system, RAPD-PCR, P-galactosidase assay
Introduction
The nomads of Mongolia produce various kinds of traditional fermented dairy products. Traditional fermented dairy products play an important role in the Mongolian diet because of their nutrient richness and medicinal potential [1, 2]. Airag is a mildly alcoholic, sour-tasting fermented drink that is usually made from the raw milk of mares; it is called Koumiss in Kazakhstan, Kyrgyzstan and Russia and Chigee in Inner Mongolia (China). Koumiss is used in treating chronic diseases of internal organs such as stomach and liver. It has high curing quality.Additionally, it supports coronary activity, reduces high blood pressure and serves as an antidote to poisoning even byradioactive substances [3,4, 5]. Khoormog is a traditional fermented mild alcoholic beverage made from raw camel milk. Khoormog is thicker than airag (koumiss), doesnot produce any casein, is quite aerated and has sweet and sour taste. It stimulatesdigestion and coronary activities, recuperates weakness and cures chronic diseases of internal organs, such as the stomach, liver and kidney and also reduces swelling [6, 7].
Therefore, both products have unique microbial compositions depending on the individual houses in which they are prepared. The end products of lactic and alcoholic fermentation are important contributors to the typical flavor and refreshing taste of these products [8]. The production of acids and antimicrobial components during fermentation may promote or improve their-Table 1. Morphological features
beneficial health effects. These characteristics are attributed to various interactions among yeast, lactic acid bacteria, and the secondary bacterialflora. The yeasts, as a part of the culture, contribute to the fermentation by supporting the starter [9]. We studied the microbio-logicaland molecular biological aspects of airag (koumiss) and khoormog; screened for fermentative organisms, and isolated and identified the yeasts from these products.
Materials and methods
For enumeration and isolation of yeastwe chose the method used bySudun Bai Yu et al. 2010 [10]. For isolation of genomic DNAwe followedstandard DNA extraction protocol using CTAB bufferfor description of Cold Spring Harbor protocols, 2009 (doi:10.1101/pdb.rec 11984).
For ITS region and RAPD-PCR amplification we followed the methods of T M. PRYCE et al. (2003) andPrashant Kumar Lathar et al. (2010) [11, 12].
Results
During this study, we collected 14 samples from-fourprovinces of Mongolia. Samples from khoormog were numbered 9 and 20 (Uvs province); 10, 11(Uvurhangai province); TS26 (Umnugovy province). Samples from koumiss originated from Tuv province and were numbered 13-19.Some morphological features of our yeast isolates are shown in Table 1.
Features Yeast cultures/isolates
TS26 776 9 10 11 12 13 14-18 19 20
Colonyform Rd Rd Rd Rd Rd Rd Rd Rd Rd Rd
Colonycolor Wh Wh Wh Wh Wh Pk Wh Wh Wh Wh
Cellshape Ov Ov Rd Ov Ov Rd Ov Ov Ov Rd
Ascospores - - - - - - - - - -
Glucose 15% + + + ++ ++ + ++ + + ++
Glucose 20% - - - - - - - - -
NaCl10% + + + + ++ + + + + ++
NaCl15% - - - - - - - - -
Colony size, 48h (mm) 2 4 6 2.5 3 2 1,5 3-5 3 5
-= negative, +=positive, Rd-round, Pk-pink, Wh-white, Ov-oval
Two of our yeast isolates (11, TS26) and one commercially available yeast strain (776-Saccharomyces cerevisiaefor positive control) were chosen for identification by API 20C AUX microtube system, a commercial product of bioMerieux Corporate. The API 20C AUX contains 19 carbon assimilation tests incubated at 30oC and read manually for turbidity. Results of reactions were visually examined at 72 h and determined to
be positive or negative based on the presence or absence of turbidity in the carbohydrate wells (Table 2). A seven-digit biocode was generated on the basis of these observations by assigning a weighted score to positive reactions. Identification was performed using the analytic profile index and a software database APILAB (Table 3).
Table 2.Result of 19 carbon assimilation
19 carbon
Isolates
£ >h о <c о h h n Pi о о j о i и ч n
d d a £ Я о £ O m i ¡zn w о <c h-1 < m R h <
11 + - - + - + - - - + + + -
TS26 + - - - + - - + - + - - - - - + + - -
776 + - - - - - - + - - - - - - + + + - +
Table 3.Identification by APILAB software
Isolates Match Identity, %
11 Candida lusitanie 73,5
TS26 Candida kefyr 98
776 Saccharomyces cerevisiae 93,5
The most reliable methods for identification of yeast strains are molecular biological sequence analysis. One molecular biological method to identify yeasts is sequence analysis of internal transcribed spacer regions with 5.8S ribosomal DNA (rDNA) gene sequence. The ITS1-5.8S- ITS2 regions of three samples (11, TS26, 776) were amplified by PCR using universal primers ITS1(5' -TCCGTAGGTGAACCTGCGG-3')
and ITS4 (5'-TCCTCCGCTTATTGATATGC-3'). Sequence similarity was analyzed using the BLAST database from GenBank (http://www.ncbi.nlm.nih.gov/BLAST/). The sequence similarity results were not the same as API 20C AUX system results (Table 4). Only one of three yeast strain performed the same identity. API 20C AUX system misidentified two or four samples.
Table 4.API 20C AUX vs. ITS sequence similarity
Yeast API 20C AUX ITS1-5.8S-ITS2
11 Candida lusitanie(73.5%) Kluyveromyces marxianus (99% FJ838773)
TS26 Candida kefyr(98) Galactomyces geotrichum (99% JQ668729)
776 Saccharomyces cerevisiae(93,5) Saccharomyces cerevisiae (100% KF447149.1)
After the misidentification of the API 20C AUX in the further identification of other yeast samples from system, we used only the sequence analysis ITS1-5.8S- our isolates (Figure 1). ITS2 region amplified by universal ITS1/ITS4 primers
Figure 1.ITS1-5.8-ITS2 regions were amplified by PCR using ITS1/ITS4 primers
In order to discriminate the same yeast strain from our isolates we used Random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR). We employed RAPD-PCR assess the genetic variability and phylogenetic relationship among yeast isolates.
Two decamer RAPD primers with 60% (OPA7; 5'-GAAACGGGTG-3') and 70% (OPB19; 5'-ACCCCCGAAG-3') GC content were selected and screened on pooled DNA of all yeast isolates (Figure 2).
M 776 Kh. TS26 9 10 11 12 13 14 15 16 17 18 19 20
200001 lOOOOl 7000|
I»
30001
Figure 2.Amplification of genomic DNA of various yeast isolates by PCR using RAPD primer.
As positive control we used Pich: Pichia pastoris, Kly: Klyveromyces lactis. 776: Saccharomyces cere-visiae, Our isolates: TS26, 9-20 A. PCR by OPA7 primer, B. PCR byOPB19 primer
RAPD-PCR primers gave distinctly reproducible and polymorphic bands in most yeast genomic DNA
samples. When we compared two RAPD-PCR results it became obvious that there was no difference between samples 14-18.The sequencing reaction of ITS1-5.8S-region of samples 14-18 was performed only on two of them (14 and 15). Among the ITS PCR products of 12 yeast isolates, nine isolates have been sequenced and identified by BLAST sequence similarity (Table 5).
Table 5.Sequence similarity of ITS1-5.8S-ITS2 region
Yeast isolates NCBI Genbank number Identity, %
TS26 Galactomyces geotrichum JQ668729.1 99
9 Cryptococcus sp. KU350382 100
10 Kluyveromyces marxianus KC544505 99
11 Kluyveromyces lactis KF646200 99
12 Rhodotorulla mucilaginosa KY104880 99
13 Saccharomyces cerevisiae KM029995 96
14,15(16-18) Saccharomyces cerevisiae CP006441.1 99
19 Saccharomyces cerevisiae KF646202 99
20 Candida zeylaoides KY102543 100
Lactose digesting ability of yeast isolates was tested by X-gal, Magenta-gal and brome-cresol-purple assay (Figure 3). Only two of 12 isolates (10, 11) were positively confirmed to have P-galactosidase activity
based on change of color. These yeast isolates were identified as Kluyveromyces marxianus and Kluyvero-myces lactis.
Figure 3. P-galactosidase assays on plate. A. X-gal and glucose containing plate; B. X-gal and lactose containing plate; C. Magenta-gal and lactose containing plate; D. Brome-cresol-purple containing plate
Conclusions
Identification of yeast by API 20C AUX system, based on 19 carbon resources assimilating ability, was effective in our case. The API 20C AUX system was the easiest and fastest method, however, it was not cheap. Later, we used only sequence analysis on theITS1-5.8S-ITS2 region. Sequence variability of this region is most reliable for identification of yeast on a species level. Our results aligned with the findings of others [13, 14], confirming that molecular methods have good potential to provide reliable, accurate species identification.
Using RAPD-PCR method by two different decamer primers we found that genomic DNA of five isolates were identical to each other, meaning that these five colonies were isolated from the same yeast strain.
Three different Saccharomyces cerevisiae(isolate numbers: 13, 14-18, 19) strains were isolated and identified from airag (koumiss) by sequencing analysis. Galactomyces geotrichum, Cryptococcus sp., Kluyve-romyces marxianus, Kluyveromyces lactis, Rhodotorulla mucilaginosa, Candida zeylaoidessix yeast species were isolated and identified from samples of khoormog.
In this study, we detected thatp-galactosidase enzyme was produced by two of our isolates (Kluyvero-myces marxianus, Kluyveromyces lactis) using a synthetic medium containing allolactoses (X-gal, Magenta-gal). Lactose digesting activity is important for utilizing whey, which is essentially a byproduct of cottage cheese making. Currently, every year several hundred tons of whey are unutilized and get discarded into the wastewater system in Mongolia. Whey contains lactose and whey proteins. In the future, we would like to use our yeast isolates in the utilization of whey at an industrial scale. All of our yeast isolates could survive high saline and glucose concentration, and did not produce ascospore. These features are basic requirement of industrial yeast cultures.
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ВЛИЯНИЕ ВРЕМЕНИ ИНКУБАЦИИ СТАРТОВОЙ КУЛЬТУРЫ СВЕРХПРОЦУЦЕНТА ЛИЗИНА CORYNEBACTERIUM GLUTAMICUM B -_11167 НА ВЫХОД ПРОДУКТА В ТЕХНОЛОГИЧЕСКОМ ЦИКЛЕ_
Роганина Виктория Юрьевна
Бакалавр, «Белгородский государственный национальный исследовательский университет»,
г. Белгород.
АННОТАЦИЯ
Цель работы - оптимизировать технологию биосинтеза лизина путем подбора параметра времени культивирования стартовой культуры. В эксперименте сокращение периода выращивания стартовой культуры с 18 до 12 часов (на 33,3 %) привело к снижению конечной оптической плотности продуцирующей культуры, и, соответственно, выхода лизина, лишь на 5,8 % в сравнении с исходной технологией. Таким образом, нами продемонстрирована возможность существенной экономии времени при минимальном снижении выхода лизина в ходе рабочего цикла. Полученные результаты должны послужить повышению эффективности использования существующих производственных мощностей.
Ключевые слова: биосинтез лизина, сверхпродуцент, кормовые добавки.
ABSTRACT
The aim of this work was to optimize the technology of lysine biosynthesis by selection of time parameter of starting culture cultivation. In the experiment starting culture cultivation time reduction from 18 h to 12 h (by 33.3 %) led to decrease of the final optical density of producing culture and respectively of lysine production only by 5.8 % compared to basic technology. Thus, possibility to save essential amount of time was demonstrated experimentally, with only minimal lowering of lysine yield during an operation cycle. Results obtained would serve to increase effectiveness of use of existing production facilities.
Keywords: lysine biosynthesis, overproducer, fodder additives.
Введение. В последнее время в Российской Федерации идет бурное развитие сельского хозяйства. Вследствие этого необходимо обеспечить животных полноценными кормами. Одним из важных компонентов является лизин. Это незаменимая аминокислота, которая не синтезируется в организме человека и животных, недостаток которой приводит к резкому снижению продуктивности животных, ухудшает качество продукции, нарушает баланс других аминокислот в синтезе белка [4].
Лизин применяют в качестве кормовой добавки. Это связано с низким его содержанием в растительных кормах и высокой потребностью в нем. Использование лизина в качестве кормовой добавки позволяет увеличить привес животных и птицы на 10 - 30 %, повышает надои молока на 12 %, увеличивает яйценоскость кур на 10 % [1].
В настоящее время для производства кормового лизина успешно используется генетически модифицированный штамм-сверхпродуцент Corynebacterium glutamicum B - 11167. Технологический цикл включает ряд этапов культивирования клеток сверхпродуцента, в том числе выращивание стартовой культуры, используемой в качестве ино-кулята при посеве основной продуцирующей культуры, обеспечивающей накопление лизина в куль-туральной жидкости [2]. В настоящем исследовании мы сосредоточились на оптимизации этапа
стартовой культуры, поставив целью подбор оптимального времени её культивирования.
Объект и методика. Данное исследование основывается на подборе оптимального времени культивирования стартовой культуры сверхпродуцента лизина С. glutamicum B - 11167 для повышения эффективности производства.
Цель исследования: оптимизировать технологию подготовки инокулята для посева продуцирующей культуры путем сокращения времени выращивания стартовой культуры без существенных потерь в конечном продукте.
В качестве объекта исследования использовали С. glutamicum штамм В - 11167 и технологию подготовки инокулята для биосинтеза лизина.
Культурально-морфологические характеристики С. glutamicum: клетки неподвижные, палочковидной формы с булавовидными вздутиями, по окраске по Грамму являются грамположитель-ными, не спорообразующие. Через 2 - 4 суток роста, на твердой агаризованной среде LB образуют колонии диаметром 2 - 4 мм кремово - желтого цвета, поверхность гладкая, форма выпуклая, край ровный, структура тестообразная, однородная [3].
В ходе эксперимента мы сравнивали три варианта с различными значениями параметра времени выращивания стартовой культуры:
