Mutant lactobacillus streptococcus thermophilic containing non-phosphorylated lactose permease

FIELD: medicine.

SUBSTANCE: invention refers to a method of producing a mutant lactobacillus Streptococcus thermophilus, to a milk ferment, a method of producing a fermented milk product and to the fermented milk product. The offered invention can be used for producing the fermented milk products with improved storage characteristics. A method of producing the mutant lactobacillus Streptococcus thermophilus characterised by weaker postoxidation, than a parent strain is implemented by introducing in DNA a genome of said parent strain of mutation codon 552 coding histidine, of teh domain HA of lactose permease. Said mutation induces replacement of said histidine by amino acid which is distinct from serine, tyrosine, histidine and threonine.

EFFECT: invention allows producing the mutant lactobacillus Streptococcus thermophilus characterised by weaker postoxidation and suitable particularly for producing the fermented milk products.

10 cl, 5 dwg, 3 tbl, 2 ex


The present invention relates to the field of dairy fermentation. More specifically this invention relates to novel mutants of Streptococcus thermophilus expressing mutant lakesuperior, transport activity which is modified. These strains and enzymes that contain them, can be used to obtain a fermented dairy products with improved properties storage.

Yoghurts are usually obtained by fermentation of milk with the help of the Association of various dairy bacteria selected from strains of Streptococcus thermophilus and Lactobacillus bulgaricus. In the fermentation process, which is carried out at a temperature of about 40-45°C, these bacteria use lactose mainly as an energy substrate and produce lactic acid that causes coagulation of milk; when the pH reaches a value of about 4.8 to 4.5, this stage of the fermentation (also called "oxidation") is stopped by cooling the product. The latter then stand in the cold during the manufacturing process and storage prior to its use.

However, cooling is not fully stops lactic fermentation; even if the product temperature is maintained at 4°C, over time there has been a gradual increase in its acidity.

This phenomenon, known as postcolony, causes the deterioration in the e organoleptic characteristics of the product during its storage.

Pustakalaya is essentially the result of the presence through bacteria residual lactose in the product at the end of stage controlled oxidation. In order to avoid it, it was proposed to use the dairy strains of bacteria fermentation or fermenting very little lactose.

Enzymes, which are important for the fermentation of lactose in Streptococcus thermophilus and Lactobacillus bulgaricus, are encoded by the operon lactose, which contains the lacS gene encoding lakesuperior, and lacZ gene, encoding β-galactosidase. These proteins, respectively, are responsible for the transport and hydrolysis of lactose. It was thus proposed to obtain nepoluchitsya of dairy strains of bacteria to obtain artificial variants or selekcionirovat natural mutants in which the activity of at least one of these enzymes is suppressed.

Patent EP 1078074 (Compagnie Gervais Danone) refers to the mutants L.Bulgaricus with suppressed activity of β-galactosidase containing antisense mutation in at least one of the genes of the lactose operon. This patent is more specifically described mutant, sequence analysis which shows two point mutations: one introduces a termination codon in the gene of β-galactosidase, which induces the inability of this mutant to use lactose; the other mutation induces a change in the am is necessaty on the level of the gene of permease (Lys-> Ans in position 122); EP 1078074 not specified the effect of this mutation on the phenotype of the mutant.

In WO 0188150 described mutants of Lactobacillus. These mutants are unable to use lactose, but retain the ability to Express β-galactosidase. In WO 0188150 does not specify the nature or the position of the mutation in question, but only that it may be in one of the structural genes of the lactose operon, for example, in permease, or plot the regulation of operan lactose, or in a gene involved in controlling the expression of the lactose operon.

Shared by mutants described in the above documents, is the complete inability to use lactose. They can grow in milk, only if it added another sugar, non-lactose, usually glucose. Properties oxidation and postcolony these mutants are controlled by the amount of added glucose.

To detect alternative to these mutants, the applicants have considered the possibility of obtaining dairy strains of bacteria that have, on the one hand, the ability to use lactose in the process of their growth, comparable to the ability of wild strains, and on the other hand, the limited ability to use lactose in the stationary phase to reduce or eliminate the effects of postcolony. To this end, they are interested in without ystavat the regulation of the transport of lactose in the mammary cells of bacteria and in particular, in cells S.Thermophilus.

The extracellular transport of lactose into the cell S.Thermophilus through lactatemia LacS. This transport lactose is a proton to the import or antepartum using extracellular galactose in the decomposition of lactose.

The transport of lactose depends on two phenomena: on the one hand, the state of phosphorylation of lactatemia and, on the other hand, the expression of lacS gene encoding this lakesuperior. Both aspects are described in detail below.

Phosphorylation of lactatemia

Protein LacS consists of a translocation domain and a domain regulation (IIA). These domains contain different his-tag residues, phosphorylation of which is involved in regulation of the transport of lactose. In particular, the domain IIA fosfauriliruetsa on histidine 552. This phosphorylation is carried out by protein HPr (histidine-containing phosphocarrier protein), which pre-phosphorylated.

HPr can be phosphorylated:

on serine ATP-dependent protein kinase; the reverse reaction of hydrolysis of HPr(Ser-P) catalizes phosphatase activity (HPr(Ser-P) phosphatase);

the histidine HPr(His-P) phosphoryl group, originating from phosphoenolpyruvate and through enzyme I(EI).

Only the form of HPr phosphorylated on histidine allows fosforilirovanii lakesuperior on g is Stidio 552.

In models in vitro with proteoliposome into the membrane surrounded by the LacS protein and its phosphorylation by HPr(His-P), it was found that this phosphorylation does not affect import lactose-proton (Gunnewijk and Poolman, 2000), and increases by about a factor of 2 the flow of lactose/galactose.

Transcription of the gene lacS

Transcription of the lactose operon is induced by growth in lactose medium. The promoter of genes lacS and Z contains the website cre (catabolite responsive element), which provides regulation by means of SSR: SSR suppresses the expression of lacS and lacZ. SSRA, in contrast, activates the transcription of the gene encoding lactate dehydrogenase (van den Bogaard et al., 2000).

The form of HPr(Ser-P) is able to interact with SRA. Together, these proteins provide the formation of the complex with cre website, resulting in repression of transcription of the gene lacS (Jones et al., 1997).

It was noted (Gunnewijk and Poolman, 2000b)that the form of HPr(Ser-P) is dominant at the beginning of exponential growth phase cultures S.thrmophilus and during the latter decreases, whereas the form of HPr(His-P) appears during the exponential phase and reaches its maximum when entering stationary phase. The transition from HPr(Ser-P) to HPr(His-P) occurs in parallel with the reduction of the lactose content and increased levels of galactose in the culture medium and a very substantial increase in the Express and lactatemia.

Thus the state of phosphorylation of the protein HPr obviously plays a role in regulating the transport of lactose, compensating for the reduction in the content of lactose in the environment through, on the one hand, the level of expression of protein lacS and, on the other hand, the regulation of its activity.

Based on the observations above, Gunnewijk and Poolman proposed the following model: if lactose is contained in the environment in an excessive amount, the lacS gene expression is inhibited by the complex HPr(Ser-P)/CcpA. During fermentation the accumulation of galactose in the medium and the decrease of free lactose leads to a reduction in the ability of bacteria to cause penetration of lactose (and, consequently, the lowering of the oxidation environment). This decrease causes a decrease in glycolytic activity and the decline in the concentration of ATP and increasing concentrations of inorganic phosphate, causing a reduction in the activity of HPr(Ser-P)kinase in favour of activity of HPr(Ser-P)phosphatase, resulting in a reduced concentration of HPr(Ser-P). This reduced concentration of HPr(Ser-P) allows to increase the catabolic repression of the gene lacS and, consequently, to increase the production of lactatemia. Parallel to the increase in HPr(His-P) will strengthen the phosphorylation of lactatemia on histidine 552 and, consequently, the ability to antiporta lactose galactose.

This model, showing that phosphorylase is the W of lactatemia on histidine 552 using HPr(His-P) increases the flow of lactose in the cell, when the amount of substrate in the medium decreases, suggests that oxidation at the end of exponential phase could slow down, if this phosphorylation to prevent. However, it is partially based on in vitro experiments and does not allow to judge about the real contribution of in vivo amplification of phosphorylation of lactatemia compared to increase its expression in the inflow of external lactose in vivo. Additionally, observations concerning the influence of the concentration of HPr(Ser-P) and HPr(His-P) on the increased expression and phosphorylation of LacS were conducted on the bacteria in exponential phase or early stationary phase; not provided any information regarding the concentrations of these two forms of HPr in the later stages of the stationary phase.

The only information about the impact of the lack of phosphorylation of LacS by histidine 552 presents only publication Poolman et al. (Poolman et al., 1992), which describes various plasmid containing the sequence encoding the enzyme LacS Streptococcus thermophilus, mutated at various residues of histidine. These plasmids were used to transform E.coli strain in which the endogenous gene lacS was previously deleterows. The transport of lactose in strains transformed various mutants was assessed by comparison, delivery, observed in the same strain of E. coli containing a plasmid coderush the th enzyme wild-type LacS Streptococcus thermophilus. No significant difference was observed in regard to mutant H552R in which native histidine is replaced by arginine. Poolman et al. bind this result or the lack of phosphorylation of the wild enzyme LacS Streptococcus thermophilus by HPr(His-P) E. coli or with the fact that this phosphorylation does not perform the functions of transport of lactose.

The authors nevertheless sought the answer to the question whether the mutation prior to phosphorylation of LacS for the remainder of histidine, to influence the properties of oxidation and postcolony mutant bacteria.

They were chosen for this study an industrial strain of Streptococcus thermophilus. This strain deposited in the CNCM 12.12.2002 under the number I-2967, allows to obtain dairy products with interesting texture; however, this strain leads to a significant postcolony.

The authors obtained and characterized a mutant of this strain, expressing instead of wild lactatemia mutant lactose permease, defosfauriliruetsa on histidine 552.

They stated that this mutant strain has a curve oxidation, which differs from the curve of the parent strain. Oxidation starts slower in the case of the mutant than the parent strain, and the maximum oxidation rate of the mutant is less high. However, the equivalent value of pH is reached after 6 hours from the beginning Fe is documentation for both strains. The most clear distinction between these two strains can be traced at the level of postcolony. Under the same storage conditions (28 days storage at 10°C), Δ (the difference between the pH at zero day and pH in 28-day) is of the order of 0.6 in the case of the parent strain and about 0.4 in the case of the mutant strain. This difference in postcolony is not due to different survival rates of bacteria. The latter is really the same for both strains. In addition, fermented products obtained using mutant have the same quality textures that obtained using the parent strain.

Thus, first of all, the present invention relates to a method for producing a mutant strain of lactic bacteria with weaker postcolonial than the parent strain from which it evolved, characterized in that the DNA of the genome, in particular in the chromosomal DNA of the specified parent strain, enter the mutation of the codon encoding histidine, fosforilirovanii by HPr(His-P) domain IIA lactatemia, and this mutation induces the replacement of the specified histidine defosfauriliruetsa amino acid.

In accordance with a preferred embodiment of the present invention the specified strain is a strain of Streptococcus thermophilus, and this mutation induces the Deputy is the histidine at position 552 lactatemia defosfauriliruetsa amino acid.

Specified defosfauriliruetsa amino acid may be any amino acid except serine, tyrosine, histidine and threonine. Preferably choose alanine.

Mainly codon encoding histidine at position 552 of lactose, permeate, replaced by a codon coding for alanine. This mutation generates a BstUI restriction site, which facilitates sorting the obtained mutants.

The method according to the invention can be achieved by the use of traditional technologies managed mutagenesis are well known to the specialist, in particular, mutagenesis through PCR.

Mutant DNA obtained in this way is then introduced into the vector, which allows to integrate the gene into the chromosome of the bacterium. Preferably this integration is carried out by recombination of insertion, which is a vector with homologous region of the bacterial chromosome.

Typically, the mutant DNA is injected into the integrative vector carrying a marker selection (for example, a gene resistant to antibiotics), and introducing the vector in bacteria, in which I want to spend mutation. The latter are then cultivated on selective medium (for example, if the token selection is the gene, are resistant to the antibiotic in the presence of the corresponding antibiotic) and Recuperat bacteria can grow in these conditions, which is the bacteria in to the e integrated vector by homologous recombination between the insert and the homologous region of a bacterial chromosome. Structure, integrated into the chromosome consists of a sequence of vector, flanked on one side by the mutant sequence, originating from the insert, and on the other hand homologous sequence of a bacterium of the owner.

Selected bacteria were cultured on non-selective medium in order to allow deletion of sequences originating from the vector, which is carried out by homologous recombination between the regions flanking these sequences. Half of the bacteria in which there was such a recombination, contains a "wild type" sequence derived from bacteria of the host, and the other half contains the mutant sequence, originating from the insert. Bacteria containing the mutation, then select any suitable way. For example, if the mutation creates a restriction site, a selection can be made on the basis of the presence of the restriction site in the amplification product by PCR mutant region.

Integrative vectors are found in many dairy bacteria. Usually for this type of bacteria integrative vector is a vector that can be entered in bacteria of this species, but which is unable to replicate in them.

As examples of vectors suitable as integrative vectors in Streptococus thermophilus, you can call Pgem5, Puc19 (Mollet et al., 1993), Pnd324 (Duan et al., 1999).

Mainly to increase the efficiency of transformation can be used as integrative vector vector with a conditional replication in selected bacteria. In this case, the bacteria, which was introduced in the vector at the first stage is cultivated under conditions favorable for replication, which allows him to settle in these transformed bacteria; in the second stage, the bacteria are cultured under conditions not favorable for replication of the vector, and, as in the case of traditional integrative vector can be selected bacteria, the chromosome where the vector was integrated.

As examples of vectors that are caused by replication, suitable as integrative vectors in most dairy bacteria, can be called the sensing vectors, described by BISWAS et al., and MAGUIN et al. (Biswas et al., 1993, Maguin et al., 1996), as well as in the PCT application WO 93/18164, or vectors pwv01 (Law et al., 1995) and Puc122 (Frere et al., 1998).

The object of the invention is also a strain of dairy bacteria, which can be obtained by the method according to the invention.

This strain differs in its chromosomal DNA contains a mutation of the codon encoding fosforilirovanii histidine by HPr(His-P) domain IIA lactatemia, and this mutation induces the replacement specified the CSOs histidine defosfauriliruetsa amino acid.

In accordance with a preferred variant of the present invention the specified strain is a strain of Streptococcus thermophilus xyla gene of lactatemia contains a mutation that causes the replacement of histidine at position 552 protein defosfauriliruetsa amino acid.

Strain milk bacteria according to the invention was deposited according to the Budapest Treaty on may 10, 2004, at the CNCM (national collection of microorganism cultures), Paris, Ul. Dr. Roux, 25, under number I-3213. We are talking about mutant strain S.thermophilus, which is a derivative of strain CNCM I-2967 (deposited at the CNCM on 12 December 2002) by entering through a managed mutagenesis mutation that replaces the histidine codon 552 by alanine codon.

The dairy strains of bacteria according to the invention during the phase of its growth have activity lactatemia like activity of the parent strain from which they originated. Thus, they have the properties the assimilation of lactose and oxidation, comparable with the properties of the parent strain from which they originated. But their activity lactatemia during stationary phase is lower than the activity of the parent strain, due to which postcolonial decreases.

Mainly dairy strains of bacteria according to the invention originate from dairy bacteria possessing the activity of β-galactosidase, and with ranaut this activity. They can normally grow in milk without the addition of other sugars in addition to lactose.

Preferably the dairy strains of bacteria according to the invention are mutants suitable for use in the food industry (or mutants “food-grade”). Preferably they are derived is characterized strains of bacteria, with preferred properties of fermented dairy products.

The present invention relates to lactic ferment containing at least one strain of bacteria, such as described above. In accordance with a private manner, lactic starter culture according to the invention contains at least one mutant strain S.Thermophilus, expressing lakesuperior, in which the histidine 552 was replaced defosfauriliruetsa balance, in combination with at least another strain of lactic bacteria, for example, strain l.bulgaricus, which also may have reduced postcolonial (for example, by mutation according to the invention lactatemia or by mutation, inactivating β-galactosidase).

A method of obtaining a fermented dairy product containing phase, during which the fermented milk with the help of lactic starter culture, such as described above, is also an integral part of the present invention, as well as any Fe is montirovannyh dairy product, which can be obtained in this way, such as yogurt, fermented milk, fermented drink, kefir, cheese or fermented milk for baby food.

Experimental examples below, illustrated by the figures in more detail some aspects of the present invention without limiting it.

Description of figures

Figure 1: comparison of the lacS gene variant I-3213 sequence of the gene lacS parent strain I-2967.

Figure 2: comparison of the curves of oxidation obtained with a mutant I-3213 and with the parent strain I-2967.

Figure 3: comparison of the rate of oxidation of mutant I-3213 and the parent strain I-2967.

Figure 4: observation of the Dornic acidity during storage of the products. Comparison of mutant I-3213 and the parent strain I-2967.

Figure 5: measurement of the viscosity η of the product.


Example 1: Obtaining mutant

The original strain is a strain of S.Thermophilus I-2967 deposited in the CNCM December 12, 2002

Gene sequence lacS histidine codon 552 (fosforilirovanii histidine) was replaced by alanine codon by double recombination. In addition, a mutation (replacement of the second nucleotide of codon 552 cytosine instead of adenine) was created in gene restriction site BstU1. This allowed selects viravati clones integrated targeted mutation in the gene lacS.

The stability of the mutation was verified by one of the resulting clones (deposited at the CNCM on may 10, 2004 under the number I-3213) by 6 serial passages followed by sequencing of the gene LacS. Figure 1 shows a comparison of the LacS gene variant I-3213 (SEQ ID NO:2) sequence of the gene LacS parent strain I-2967 (SEQ ID NO:10. The presence of mutations is obvious: now histidine codon 552 encodes alanine. Other unwanted mutations in the gene LacS no.

Mutant I-3213 suitable for use in the agri-food industry, as it does not contain any residual sequence of the plasmid used to integrate mutations in the gene LacS.

Example 2: Physiological tests performed with a mutant I-3213

In order to establish that the mutant I-3213 is less postcolonial than the parent strain I-2967, received products with a pure strain and the observations were carried out to J+28: oxidation, postcolonial, survival, texture.

2.A Protocol

Enlivening strains by 2 passages

Obtain starter cultures on sterile milk with the addition of yeast extracts and incubation at 44°C to achieve the acidity 85°D (corresponds to 108, 109UFC/ml).

Sowing a mixture of 120 g of skim milk powder + 930 ml of water + 1 g peptone is latina N3 (Organotechnie), pasteurized for 10 minutes at 95°C With 1% (v/v) enzyme.

Incubation in banks in an oven at 44°C until reaching pH 4,65.

Stop fermentation by placing the cans in ice water for 30 minutes.

Storing food for 28 days in a refrigerator at 10°C.

2. Observation-Comparison of oxidation using mutants and strains I-2967.

V Curves oxidation

Using the CINAC system (Kinetics of oxidation, Alliance Instruments) pH is measured continuously over time. This way you can receive:

curve oxidation for each strain,

primary derivative with respect to time, which accelerates oxidation.

V Measuring pH

The observation of the evolution of pH during storage of products carried out with the help of pH meter Mr 220 company Mettler Toledo.

V Measurement Dornic acidity

The measurement of the Dornic acidity (D°) allows you to chitravati the molar concentration of ions N3About+. The number of degrees Dornic corresponds to the number in the tenths of a millimeter of solution of sodium hydroxide at a concentration of 0.1 N, required to neutralize 10,32 g of milk. Neutrality visualize the color of the indicator, phenolphthalein. In range (pH 8,2) of colorless phenolphthalein turns pink. Degree Dorni corresponds to 100 mg of lactic acid per litre of milk.

W. The mixture to test products

Dairy environment is obtained from 120 g of skim milk powder (Milex 240, Arla Food Ingredients)+930 ml permuteran water + 1 g peptide N3 (Vital Armor 950, Armor Protéines). The mixture is stirred until complete homogenization. Then Wednesday again hydronaut for 30 minutes at room temperature, then pasteurized at 95°C for 10 minutes.

V Survival of strains

Measuring the survival of strains is carried out in a medium containing agar-agar M17 and sucrose. Isolated on the surface by means of helical device (ensemenceur spirale)(WASP firm AES). Incubated at 37°C in the presence of N2WITH2. Read after 72 hours from the beginning of incubation.

Curves oxidation in pure strains are constructed in two instances.

V Interpretation

The results, shown in figure 2 and 3 show that the curve oxidation mutant I-3213 smoother curve than the parent strain, with a more pronounced effect of urea and that the maximum rate of oxidation below. However, pH 4,50 both strains is achieved by 6 hours.

2. Observation-Comparison of postcollege using a mutant I-3213 and strain I-2967

Figure 4 shows the results of observation of the Dornic acidity during storage of the products by comparing products derived f is ramentacea using a mutant I-3213 and the parent strain I-2967.

The results of pH measurement directly after stopping the fermentation (J0) and after 28 days of storage at 10°C are presented below in table 1.

Table 1
pH J0pH J28
I 2967a4,654,07
I 2967bwith 4.644,08
I 3213b4,654,19

The combination of these results confirms that the mutant has a lower postcolonial than the parent strain.

2.D - Monitoring-Comparison of survival of mutants and strain I-2967

In the following table 2 shows the number of colonies of bacteria present in 1 ml of the product after 28 days of storage at 10°C.

Table 2
UFC/ml J28
I 2967a1,4 .107
I 2967b7,6 .107
I-3213a7,4 .107
I 3213b1,0 .108

These results show that after 28 days of storage after stopping the fermentation mutants have the same high survival rate as the parent strain.

E - Monitoring-Comparison of the texture of the products obtained using the mutant, and products obtained by means of the parent strain I-2967

Spend measure the texture of the products of the same series production. All the measurements were carried out in triplicate (3 banks for withdrawal of the same dimension).

To measure the texture of products J+7 used three methods of measurement:

The measurement penetrometry using TAXT2 (10°C).

Measurement of flow after mixing by hand using Rhéomat 260 (4°C).

Viscosity measurement in serum after centrifugation at MCR300 (20°C).

These different measurement methods textures are described in detail below.

E - viscosity Measurement in serum of solid products

The interest of this method lies in the analysis savored the solid products in order to confirm the rheological characteristics of milk, fermented with strains.

This method of analysis allows the first stage to recover the serum solids. For this amount of product, approximately 50 g, centrifuged at 631 g for 10 minutes at room temperature, so you can collect the serum present in the gel dairy products, fermented by strains. Then the serum is recovered and subjected to the same centrifugation in order to remove the greater part of product residue. The remainder of these particles is deposited and forms a brittle residue.

Then the serum viscosity is measured at 20°C and a fixed gradient shift 100s-1within a minute. Three measurements were carried out also on three banks with milk fermented with the same strain in the same conditions. The device used for this analysis represents a rheometer (Anton Paar Physica® MCR 300, equipped with a coaxial geometry with a double gap type DG 26.7/TEZ 150 R-C and system temperature control with Peltier effect. This swivel allows to estimate the viscosity of the serum with a constant gradient shift by one point per second.

Usually the first two values of this dimension are inconsistent and distorted initialization rotary system. Therefore, the viscosity of each serum [Vs] define p is the average calculated using the values measured by the device, except the first two.

E - Measurement penetrometry (Fgel-Dgel-F15vv)

The device used for this measurement is Thermo Rhéo TAXT2 (Anton Peer Physica, Austria).

A cylinder with a diameter of approximately 1 cm penetrates into the gel (at 10°C) at a constant speed of 15 mm depth. During immersion of the rolling element in the product gel and resists deformation before rupture. Measure the force produced as a result.

Have the following settings:

Fgel - power gel (g)corresponding to the amount of force exerted movable element at the moment of rupture of the gel (the first peak of the curve).

Dgel - a distance corresponding to the strength of the gel (mm), i.e. the depth to which plunges the movable element at the moment of rupture of the gel.

F15 - force at a depth of 15 mm (g)corresponding to the power measured at the stop of the movement of the moving element.

A flow-Viscosity flow

This method consists in the determination of the viscosity of the solid products after manually mixing and incubation for 30 minutes at 4°C. At 4°C was performed three measurements on three banks of milk fermented with the same strain in the same conditions. For this analysis, use viscosimeter Mettler® RM 260, cooled and supplied the military coaxial system type DIN 145. This swivel system allows us to observe the destruction of the product depending on the gradient of the linear shift, i.e. the voltage at a given gradient.

The results are obtained in the form of the curve of a continuous flow, with ascending and descending plot from 0 to 20s-1.The product undergoes the shift gradient increasing from 0 to 20s-1for 1 minute. This phase corresponds to the upward section. Then he undergoes a shift gradient, decreasing from 20 to 0 s-1for 1 minute, which corresponds to the descending section.

Each descending curve is modeled then the Casson model:

τ is the stress (PA);

τ0the threshold of the flow of product (PA) [threshold 4];

η is the viscosity (PA·s) [V4];

D - shift gradient (s-1).

This simulation to Spend with subsequent direct linear regression on the descending part of the curve allows you to set an important parameter, which is the viscosity η, corresponding to the slope of the straight regression.

Figure 5 shows how to calculate the viscosity in this model.

E Results

The results obtained by each measurement method, presented below in Table 3.

Tab the Itza 3
The serum viscosity (MPa·s)2,182,31
Fgel (g)33,5833,18
Dgel (mm)3,292,96
F 15 mm (g)39,2338,78
The viscosity of the stream11541171

Analysis of variance (P<0,05) are based on measurements of the texture (for each parameter value compare Student test):

Parameters Power gel, the Distance when the strength of the gel and the Strength of 15 mm show that the two strains did not have significant difference.

Parameter viscosity, the measured flow, shows that the two strains did not have significant difference.

The viscosity of the serum being reproduced, shows a significant difference between the two strains, but the mutant has a higher viscosity than the strain I-2967, this proves that changes flowed the tours did not happen.

E - Interpretation

Measurement of the texture of fermented products obtained using the mutant and the parent strain, allow to show that changes textures associated with the mutation has not occurred.

2.F - Conclusions

The mutant strain I-2967 with defosfauriliruetsa lactosaminated was received in two recombinations.

This mutant, named I-3213, has:

curve oxidation than the curve of the parent strain (slow rate),

less postcollege on the 28th day

texture similar to the texture of the parent strain, and

Good survival at 28 day.

1. A method of obtaining a mutant lactic bacteria of the species Streptococcus thermophilus, with weaker postcolonial than the parent strain from which they are derived, characterized in that the DNA genome of the specified parent strain introducing the mutation of codon 552 encoding histidine, fosforilirovanii by HPr(His-P) domain IIA of lactatemia, and this mutation induces the replacement of the specified histidine amino acid other than serine, tyrosine, histidine and threonine.

2. The method according to claim 1, characterized in that the parent strain is a strain of Streptococcus thermophilus, and the fact that this mutation introduces alanine codon instead of a histidine codon 552.

3. Mutant lactic bacteria of the species Streptococcus thermophilus for use in dairy starter that can be obtained by the method according to any one of claims 1 or 2.

4. Mutant lactic bacterium according to claim 3, characterized in that it has the activity of β-galactosidase.

5. Mutant lactic bacterium according to claim 4, characterized in that it is a mutant strain of S.Thermophilus, deposited on may 10, 2004, at the CNCM under number 1-3213.

6. Lactic ferment containing at least one mutant lactic bacterium according to any one of p-5.

7. Lactic starter culture according to claim 6, characterized in that it contains at least one mutant lactic bacterium according to any one of p-5, type-specific S.Thermophilus, in combination with at least one strain of L.bulgaricus.

8. A method of obtaining a fermented dairy product, comprising a stage on which the fermented milk through the dairy starter culture according to claim 6 or 7.

9. Fermented dairy product that can be obtained by the method according to item 8.

10. Fermented dairy product according to claim 9, characterized in that said product is selected from yoghurts, fermented milk, fermented beverages, yogurt, cheese and fermented milk for baby food.


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SUBSTANCE: nutrient medium contains soya flour and distilled water at preset quantitative ratios.

EFFECT: Deinococcus radiodurans yield increase.

2 tbl, 8 ex

FIELD: food industry.

SUBSTANCE: lysine-producing gram-positive bacterial strain for biologically active compounds delivery to ruminant animals is grown by way of at least single passage through the growth medium containing a quantity of lysozyme which is effective for bacteria cell walls growth induction; these bacteria are resistant to protozoal intake. The bacterial strain is extracted from the medium containing lysozyme and used for preparation of a feed supplement for ruminant animals passing through the ruminant animal paunch. The ruminant animals feeding method involves addition of an effective quantity of the feed supplements (passing through the paunch) to the feed ration.

EFFECT: increase in resistance of the lysine-producing gram-positive bacterial strain to inactivation in the paunch which strain is used for biologically active compounds delivery to ruminant animals.

17 cl, 5 dwg, 2 tbl, 13 ex

FIELD: chemistry.

SUBSTANCE: culture medium contains dry nutrient agar, glucose, 5- aminosalicylic acid, an extract of nutrient yeast, paraaminobenzoic acid, bromthymol blue, tris-buffer, sodium carbonate, brilliant green and microbiological agar.

EFFECT: invention shortens duration of identifying klebsiell.

3 ex

FIELD: medicine.

SUBSTANCE: method provides collection of an investigated material from a surface with a tampon made of an elastic finely porous material wetted in a solution inhibiting the development of other microorganisms. The collected investigated material with tampons is kept in the same solution for 18-24 hours. The investigated material is separated from a tampon and centrifuged. The precipitation is neutralised with 1% citric acid (in the ratio 1:1). No more than one-third of the investigated material is separate from the precipitation. The separated precipitation portion is placed on a phase-contrast slide to conduct phase-contrast microscopy. The residual precipitation is placed on a nutrient medium to be cultivated thereon if more exact quantitative assessment of mycobacterial pollution of the investigated surface is required.

EFFECT: invention allows reducing mycobacteria detection time.

2 tbl, 1 ex

FIELD: medicine.

SUBSTANCE: strain is obtained by insertion into genome of Escherichia coli, isolated from clinically healthy representative of canine family, of genes, determining synthesis of microcin C51, B-subunit of toxin, as well as vaccine version of elt-operon and A-subunit, enhancing immune response. Strain possesses expressed adhesion to mucous membrane of intestine of animals of canine family. Frozen dried culture of Escherichia coli EB387 represents probiotic medication for protection of animals of canine family against toxicoses, induced by cytotonic toxins of A1B5 type.

EFFECT: normalisation and stabilisation of qualitative and quantitative composition of gastrointestinal tract microflora in representatives of canine family.

2 cl, 3 ex

FIELD: medicine.

SUBSTANCE: nutritional medium contains liquid phase, content of liquid phase is determined by biological peculiarities of particular microorganism and solid phase. Solid phase contains coagulated serum and agarose in specified ratio of components.

EFFECT: invention allows to increase reliability of estimation of results of microorganism culture growth and development.

1 ex

FIELD: medicine.

SUBSTANCE: claimed is powder composition, possessing lipase activity. Composition contains filtering auxiliary material(s) and product, obtained by fine grinding of lipase, originating from Thermomyces sp., immobilised on silicon carrier(s), to average particle size 1 mcm or larger to smaller than 300 mcm. Also claimed are methods for re-etherification of fats and oils and for etherification with application of obtained lipase powder composition.

EFFECT: powder composition by the invention possesses improved lipase activity.

10 cl, 1 dwg, 2 tbl, 8 ex

FIELD: medicine.

SUBSTANCE: method of prevention and method of treatment of gastrointestinal diseases in animals or people, such as diarrhea, antibiotics-associated diarrhea, inflammatory intestinal disease and diarrhea, caused by Clostridium difficile (CDAD), includes stage of introduction of efficient amount of strain of Bacillus subtilus ATCC PTA-6737, as probiotic. Strain produces lipopeptide sufractine and represents medication for treatment of gastrointestinal diseases in animals or people, such as diarrhea, antibiotics-associated diarrhea, inflammatory intestinal disease and diarrhea, caused by Clostridium difficile (CDAD).

EFFECT: invention ensures high efficiency in treatment and prevention of gastrointestinal diseases in people or animals.

15 cl, 14 dwg, 12 tbl, 4 ex

FIELD: medicine.

SUBSTANCE: there is offered a method of engineering yeast strains - stable human growth hormone (somatotropin) producers. Also, there are offered two strains Y-3506 of Russian National Collection of Industrial Microorganisms and Y-3507 of Russian National Collection of Industrial Microorganisms - stable human somatotropin producers. The producer strains have been prepared by sequential integration of expression plasmid into a recipient stain genome. Each plasmid carries in its structure a somatotropin gene (GH1); fused with a leader sequence controlled by the GAL1 promotor, as well as one of the genes URA3, LEU2, TRP1 and HIS3 complementing auxotrophic recipient strain mutations.

EFFECT: efficacy of the produced strains is 100-130 mg of somatotropin per 1 l of the medium.

3 cl, 7 dwg, 6 ex

FIELD: medicine.

SUBSTANCE: invention relates to field of medicine and deals with anti-sense oligonucleotides for treatment and/or prevention of, at least, one of the following diseases: asthma, hypereosinophilia. Essence of the invention includes oligonucleotides aimed against sequences of nucleic acids, coding receptor, selected from group, which consists of receptor CCR3 and common subunit of receptors IL-3, IL-5 and GM-CSF with sequences SEQ ID NO:1 and SEQ ID NO:14.

EFFECT: reduction of toxicity in comparison with hormonal medications.

39 cl, 8 ex, 11 tbl, 21 dwg

FIELD: medicine.

SUBSTANCE: offered is a microorganism producing homosuccinic acid, of Mannheimia, Actinobacillus or Anaerobiospirillum genus. Said microorganism has been produced by destructing a lactate dehydrogenase (ldhA) coding gene, a phosphotransacetylase (pta) coding gene and an acetate kinase (ackA) coding gene, without destructing a pyruvate format lyase (pfl) coding gene. Besides, offered is a method for producing said microorganism and a method for producing succinic acid with applying thereof. The offered mutant microorganism exhibits a property of high growth rate and efficacy of succinic acid in the absence or low production of other organic acids, as compared to the previous strains producing succinic acid.

EFFECT: said microorganism is available for producing succinic acid for industrial application.

13 cl, 7 dwg, 2 tbl, 5 ex

FIELD: medicine.

SUBSTANCE: constructed are recombinant plasmid DNA pAUTL-GFP bearing a hybrid gene coding green fluorescent protein (GFP) and a signal sequence of autolysin from Chlamydomonas reinhardtii. The produced plasmid is used to transform an Agrobacterium strain. A suspension of the transformed agrobacterial cells in a logarithmic growth stage is incubated with cells of Chlorella microalgae for 14-16 hours A protein secretion level is evaluated by measuring fluorescence of a culture environment.

EFFECT: new compounds show effective biological properties.

2 cl, 1 dwg, 1 tbl, 2 ex

FIELD: medicine.

SUBSTANCE: invention represents recombinant plasmid DNA pBinPLUS-ARS-EPSPS sized 14.8 kbp providing gene expression of agrobacterial 5-enolpiruvoyl-shikimate-3-phosphat-synthetase under the control of a gene promoter of chlorella alpha-tubulin in transgene chlorella microalgae consisting of the following elements: DNA of vector plasmid pBinPLUS-ARS sized 12440 bp, Hindlll-BgIII of a DNA fragment sized 500 bp, containing a gene promoter of chlorella alpha-tubulin hlorelly, Bglll-Xbal of a DNA fragment sized 1610 bp, containing a coding sequence of EPSPS gene with a signal peptide, Xbal-Hindlll of a DNA fragment sized 230 bp, containing 35S terminator of cauliflower mosaic virus.

EFFECT: use of recombinant plasmid DNA pBinPLUS-ARS-EPSPS will allow to produce glyphosate-resistant transgene chlorella microalgae.

3 dwg, 2 tbl, 2 ex

FIELD: chemistry; biochemistry.

SUBSTANCE: invention relates to biotechnology and is in form of isolated monoclonal antibodies, particularly human monoclonal antibodies which specifically bond with PD-1 with high efficiency. The invention also relates to molecules of nucleic acids which code the disclosed antibodies, expressed by vectors which contain such nucleic acids, as well as to host cells containing said vector. The invention also relates to methods of increasing immunoreaction, inhibiting cell growth, as well as treatment of infectious diseases in a person using the disclosed antibodies.

EFFECT: disclosed antibodies enable to carry out immunotherapy when treating human diseases and reducing their side effects.

10 cl, 100 dwg, 8 tbl, 25 ex

FIELD: medicine.

SUBSTANCE: invention refers to recombinant insect defensin expression in a cell of filamentous fungi Aspergillus. The given cell is transformed by a genetic maker coding insect defensin, and contains one or more intron sequences. The intron sequence can be enclosed in any maker segment of nucleic acid.

EFFECT: application of the offered maker makes the level of recombinant defensin expression to be improved as compared to application of a nucleic acid maker without intron sequences.

6 cl, 1 tbl, 6 ex

FIELD: chemistry; biochemistry.

SUBSTANCE: present invention relates to a novel fluorescent protein from Entacmaea quadricolor and its functional mutants. The invention discloses nucleic acids which code the said protein, a vector which contains the said nucleic acids, a transgenic cell which carries the vector and a method of obtaining the said fluorescent proteins from transgenic cells. The composition of the said proteins and nucleic acids can be used in various applications and methods, particularly for labelling biomolecules, cells or cell organelles. The disclosed protein and nucleotide sequences can be used for testing activity of promoters under various conditions.

EFFECT: obtaining proteins with primarily red or far-red fluorescence.

7 cl, 7 dwg, 7 ex

FIELD: chemistry; biochemistry.

SUBSTANCE: invention relates to biotechnology and the DNA version related to adult hypolactasia. The substance of invention includes a nucleic acid molecule containing the 5'-end part of the intestinal digestive lactase-phlorizin hydrolase (LPH) gene which participates or serves as an indicator of adult hypolactasia. The said nucleic acid molecule is selected from a group consisting of: (a) nucleic acid molecules having the SEQ ID N0:1 sequence or containing it, where the SEQ ID NO:1 sequence and (b) nucleic acid molecules having the SEQ ID NO:2 sequence or containing it, (c) nucleic acid molecules consisting of at least 20 nucleotides whose complementary strand is hybridised in strict conditions with the nucleic acid molecule at point (a) or (b), where the said polynucleotide/or nucleic acid molecule contains a cytosine residue in a position corresponding to position -13910 in the 5'-direction from the LPH gene; and (d) nucleic acid molecules consisting of at least 20 nucleotides whose complementary strand is hybridised in strict conditions with the nucleic acid molecule at point (a) or (b), where the said polynucleotide/nucleic acid molecule contains a guanine residue in a position corresponding to position -22018 in the 5'-direction from the LPH gene.

EFFECT: design of a method of testing presence or predisposition to adult hypolactasia, which is based on SNP analysis, contained in the said nucleic acid molecule.

65 cl, 7 ex, 8 tbl, 9 dwg

FIELD: medicine, pharmaceutics.

SUBSTANCE: there is described an immunogen for making an immunogenic cancer composition free of DNA-binding function and all domains of a zinc finger, on the basis of polynucleotide coding a nonfunctional mutant form of a related molecule ("brother") of regulator of imprint sites (BORIS) of protein, polypeptide or peptide, containing amino acid sequence presented in the description. The immunogenic cancer composition contains aforementioned immunogen and an adjuvant chosen particularly from cytokine, chemokin, a costimulating molecule. There is described an expression vector containing polynucleotide, coding above-stated protein, e.g., in bacterial systems, mammal systems, in yeast or viral systems. The cancer vaccine under the invention contains polynucleotide (immunogen), additionally the adjuvant and, if necessary, a pharmaceutically acceptable carrier. The invention describes the method for of cancer immunisation of a mammal with using said immunogen on the basis of polynucleotide.

EFFECT: invention allows improving effectiveness of cancer prevention.

28 cl, 7 dwg, 2 tbl, 1 ex

FIELD: medicine.

SUBSTANCE: invention refers to biotechnology, particularly to recombinant production of human oncofetal proteins, and can be used for producing a 404 to 609 amino acid fragment of human alpha-fetoprotein. The Escherichia coli BL21(DE3)/pAFP11D3 strain - a producer of the 404 to 609 amino acid fragment of human alpha-fetoprotein is produced by Escherichia coli cell transformation of the BL21(DE3) strain of plasmid DNA pAFP11D3.

EFFECT: invention allows reducing potential human viral contamination when using a recombinant method to produce said alpha-fetoprotein fragment exhibiting affinity to an alpha-fetoprotein receptor and exposed to receptor-mediated endocytosis.

2 cl, 7 dwg