SU1409195A1 - Method of producing bacterial preparation for rennet cheeses - Google Patents

Abstract

The invention relates to the dairy industry, namely, to cheese, and is intended to suppress the development of the bacteria of the coliform group in the production of cheeses with a low second heating temperature. The purpose of the invention is to improve the quality and sanitary and hygienic indices of cheeses. The method consists in using an inoculum with a ratio between Streptococcus lactis species when preparing the preparation. Streptococcus lactis subsp. high and low acid-forming diacetilactis, Leuconcs-tos time or L.cremoris in combination with Lactobacillus plantarum, equal to 1.2:: 0.8: 0.2: 1.8: 2.0, respectively, and the composition of the microflora of the drug is used in strains that give the zone of growth of Escherichia coli when determining the perpendicular p-HIIX method of hornings on buffered (pH 6.7i iO, 2) agar with hydrolyzed milk and 0.002-0.003% molecular-clotting enzyme not less than 5 mm, Lactobacillus plantarum strains are used from milk-sour sticks. They give cultivation of milk acidity for 7 days of cultivation at 37 ° C. with milk-converting enzyme (90-130) T.4 table. 1 il. cl

Description

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The invention relates to the dairy industry, namely, to cheese making, and is intended to suppress the development of bacteria of the group of intestinal sticks in the production of cheeses with a low second heating temperature.

 The aim of the invention is to improve the quality and improvement of sanitary and hygienic indicators of cheeses with a low second heating temperature.

The composition of the proposed drug includes strains with antagonistic activity on this medium 5-6.5 mm that provides the same antagonistic activity of the finished product.

Presented in table 1 of the results of monitoring the development of OGP in Kostroma cheese, produced using starters, the microflora of which has different antagonistic activity, it is clear that the use of starter with microflora having antagonistic activity to KP 5 mm, provides not only a significant suppression of KP growth on the initial stages of cheese ripening, but also the rapid cell death of these bacteria.

In addition, milky-sour streptococci and leukoostoks are checked for the complex of features that are valuable for cheese-makers, as well as for selection to the BP-Uglich-№ 4 bakpreparat.

An important component of the microflora of the proposed drug are lactic acid sticks, since they greatly accelerate the elimination of E. coli (Fig. 1).

A comparative study of the effect of certain properties of lactic acid bacteria — antagonists of intestinal sticks — on the quality of the cheese, established a significant effect of the acid-forming activity of the lactic acid sticks on the organoleptic characteristics of the cheese. Thus, the inclusion of L.plantarura with high acid-forming activity into the composition of the starter (tabl. Was found in the appearance of sour taste and decrease in organoleptic assessment by 4 points compared with the cheese produced with a leaven containing L.plantarura with lower acid-forming activity Consequently, this property of lactic acid sticks, significantly influencing

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on the quality of the cheese, it is necessary to consider when selecting strains in the composition of drugs

Based on this, lactic acid sticks are selected by sensory evaluation of milk clots, combined with streptococci, antagonistic activity and by acid formation. The preparation includes strains that gave a zone of absence of Escherichia coli not less than 5 mm on agar with hydrolyzed milk, phosphate buffer and 0.002-0.003% of a milk-clotting enzyme preparation, and with a maximum titrated acidity of no more.

Along with the selection of cultures and their selection into the microflora of bacterial preparations, the modes of biomass accumulation of lactic acid bacteria, in particular the inoculum composition, which largely determines the composition and properties of the finished bacterium, are important in the technology of producing active bacterial preparations.

S.lactis (1.2% of the volume of biomass storage medium), S.lactis subsp. diacetilactis active acid forcing (0.8%) and weak acid forcing (0.2%), L. lactis or L. cremoris (1.8%), L.plantarura (2.0%).

Using the specified quantitative and qualitative composition of inoculum allows to obtain the optimum ratio for the production of cheese between groups of lactic acid bacteria (total amount, aromatic bacteria, sticks 360 :.

Skimmed milk is used as a medium for biomass accumulation; its proteins are hydrolyzed with proteolytic enzymes, with the addition of buffer salts and growth stimulants. Cultivation is carried out at 30-32 s for 10-12 hours

The antagonistic activity of the proposed drug is normalized and should be at least 5 mm when it is determined on agar with hydrolyzed milk, phosphate buffer, and 0.0025% of the milk-clotting drug using the well method.

The method is as follows. 1 ml of phosphate buffer with a pH of 6.8, 10 ml of molten agar with hydrolyzed milk, 1 ml of a 0.025% aqueous solution of a milk-fermenting enzyme preparation (for example, OP-VNIIMS) are introduced into a 1P cup. After hardening the medium, 10 ml are poured into the cup.

melted and cooled to 40- / s ,,

WH with semi-liquid agar with hydrolyzed milk, in which, before pouring, 1 ml of phosphate buffer was introduced into the Petri dish, 3-GB dilution of an 18-hour E. coli VKM-125 culture grown on m Szepton broth at HL C and 0.025% a solution of the milk-converting enzyme preparation. Petri dishes with double-layer agar are dried for 30 minutes. Then, in the thickness of the agar plate, dimples (wells) are cut out with a cork bore without damage to the lower layer of agar. 0.2 ml of the starter prepared from bacterial preparation in the well is transferred to the well. The plate is left for 18 hours at room temperature, then incubated at 37 ° C (18-24) hours | then the diameter of the growth inhibition zone of the test culture is measured.

The magnitude of the antagonistic activity (A) is characterized by the width of the growth inhibition zone of the test culture (mm), which is calculated by the formula

l -Sij-5,

where D, is the diameter of the zone of growth inhibition

test culture, mm; Dg - hole diameter, mm.

The method of holes allows to take into account the antagonistic activity of the mixed microflora of the drug with its growth in milk in the same ratio as in the preparation of production ferment.

Example 1. The strain S. lactis, S. lactis subsp. diaceti-lactis with high and low acid-forming rates, L. cremoris, L.plantarum by conventional methods as follows: acid-, gas-, aromatizing, relation to rennet enzyme, ability not to form bitter peptides, antagonism to mesophilic lactic acid streptococcus, phagal tolerance, relation to the combined temperature regime, lipolytic activity, organoleptic evaluation, microscopic preparation.

The strains selected according to the listed indicators are checked for antagonistic activity to the intestinal tract.

stick by the method of perpendicular strokes on agar with hydrolyzed milk, 10% phosphate buffer with pH 6.7 and 0.0025% of milk-clotting enzyme preparation OP-VNIIMS.

As a result, the following cultures are included in the composition of the bacterial preparation:

S.lactis 825 (width of the zone of stunt growth of E. coli 5.5 mm

S.lactis 273, (6 mm);

S.lactis subsp.diacetilactis with a high rate of acid formation 139 (6.5 mm) and a low rate of acid formation 875 (5 mm);

L.lactis 240 (12 mm);

Lactobacillus plantatum 28 (the width of the zone of the delay in the growth of Escherichia coli is 10 mm, the acidity of milk with 0.0025% of the enzyme preparation OP-VNIIMS after 7 days of cultivation at 37 ° C is 120 T).

Inoculum is prepared from the selected strains, for which 1% of the culture of each species is introduced into the nutrient medium (S.lactis, 0.5% of each strain, which in total amounts to 1%). The inoculum of streptococci and leukonostok is incubated at ZO C: S.lactis and S.lactis subsp. high speed acid forming diacetilactis for 16 hours; S.lactis subsp. low acid formation diacetilactis and leukoiostok for 36 hours; L.plantarum at 37 C for 24 h.

The prepared inoculum is introduced into the biomass storage medium in the following amounts (% of the volume of the medium) S S. lactis 1,2; S.lactis subsp.diacetilactis with a high rate of acid formation - 0.8 and with a low rate of acid formation - 0.2; Leuc.lactis 1.8; L.plantarum 2.0. Only 6%.

The biomass storage medium is prepared as follows.

D5 kg of skimmed milk powder is dissolved in 1000 liters of tap water, the pH is adjusted to 6.8, warmed to 55 ° C, 225 g of protosubtilin GLC enzyme with a proteolytic activity of 70 units, pre-activated is introduced into I l of water at 30 C for 12 min. Milk hydrolysis is carried out at 55 ° C for two hours.

After hydrolysis of proteins, 10 kg of citric acid, 4 kg of vitamin B, fodder, 200 g of sulfur are introduced.

acid manganese, 2 kg of acetic acid sodium, 10 kg of glucose, 100 g of sulfuric acid iron. Mix, set pH 7.4, sterilized for 15 min.

The accumulation of bacterial cells was carried out in the LKB fermenter at BF C and pH 6.8 for 1 hour with automatic deoxidation with 30% solution of NaOH.

At the end of the incubation, the medium is rapidly cooled to M C and centrifuged in an OTP supercentrifuge at 15,000 rpm.

The resulting biomass is suspended in a 1: 1 ratio with a protective medium based on hydrolyzed milk containing, g / l:

Sugar100

Ammonium citric trisubstituted 20

Ascorbic Acid 5

Sulfuric Magnesium 0.5

Apilak0,7

The bacterial suspension is poured into the drier cuvettes, frozen at minus for 3 hours, and dried under vacuum at the KS-30 sublimation unit.

The dry preparation is ground into powder, packed in glass vials and sent to the enterprises. Table 3 shows the indicators of the dry preparation.

PRI mme R 2. Select the strains S.lactis, S.lactis subsp. diacetilactis with high and low acid formation rates, Leuc. cremoris, Lactobacillus plantarura read on the following index m: acid-, gas, aromatoobrazovanie of wearing to the rennet, the ability not form bitter peptides antagonism mesophilic lactic acid streptococci, phage-resistant, the ratio to the combined temperature mode, the lipolytic organoleptic evaluation activity, microscopic preparation.

The strains selected according to these indicators are tested for antagonistic activity to Escherichia coli using the method of perpendicular strokes on hydrolyzed milk agar, 1OZ phosphate buffer with pH 6.8, and 0.0032 of the enzyme preparation OP-VNII

The composition of the bacterial preparation include the following cultures:

956

S.lactis 849g (width of the zone of stunting of E. coli 5 mm;

S.lactis 3575 (6 mm);

S.lactis subsp. diacetilactis with high acid formation rate (9 mm) and low acid formation rate (6 mm);

Leuc.cremoris 381 (13 mm);

L.plantarum 28 (the width of the zone of stunted growth of Escherichia coli is 10 mm, the acidity of milk with 0.0025% of the enzyme preparation OP-VNIIMS after 7 suc cultivation at 37 C is 1 20 tons).

Inoculum is prepared from the selected strains, for which 1% of each type of culture is introduced into the nutrient medium (S.lactis, 0.5% of each strain). Inoculum of streptococci and leukocytosis is incubated at 30 ° C: S.lactis and S. lactis subsp.diacetilactis with a high acid formation rate of 16 hours, S.lactis subsp.diacetilactis with a low acidification rate, and leukostock, 36 hours; L.plantarum at 3 hours for 24 hours.

The prepared inoculum is introduced into the biomass storage medium in the following amounts (% of the volume of the medium): S. lactis 1,2; S.lactis subsp.diacetilactis with a high acid formation rate of 0.8 and a low acid formation rate of 0.2; Leuc.cremoris 1.8; L.plantarum 2.0; Only 6%.

Further technological operations: preparation of biomass storage media, accumulation of bacterial cells, cooling, centrifuging, suspending with a protective medium (including the composition of the protective medium), freezing the bacterial suspension, freeze drying, grinding, packaging and packaging of the preparation are performed as in example 1.

Table 4 shows the indicators obtained dry the drug.

The use of the proposed bacterial preparation in the production of cheeses with a low temperature of the second heating as compared with the prototype (the bacterial preparation BP-Uglich-No. 4) makes it possible to significantly suppress the development of EHEC in cheese to improve the quality and improve the sanitary and hygienic indices of cheeses, as well as to increase the output top grade cheese by 1.95%.

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Claims (1)

Invention Formula A method for producing a bacterial preparation for small rennet cheeses, which involves the selection of cultures of mesophilic lactic acid bacteria according to production characteristics, including taking into account antagonistic activity to E. coli, inoculation of the nutrient medium with cultures of Streptococcus lactis, Streptococcus lactis subsp.diaceticetes high and low acid formation rates and Leuconostoc sp., accumulation of biomass, centrifuging it, mixing with a protective medium, lyophilic drying, characterized in that, in order to achieve the quality of the target odukta and improve the health indicators cheese cultures used in the selection of 958 strains, which give the intestinal stick growth inhibition zone during the determination by the method of perpendicular strokes on the buffered agar with hydrolyzed milk pH 6.5-6.9 and 0.002-0.0037, the milk-clotting enzyme from 5 to 6.5 ml, the composition of inoculum In addition, lactic acid sticks of Lactobacillus plantarum, which give the acidity of milk with a milk-coagulating enzyme from 90 to 130-T for 7 days, from the cultures of Leuconostoc spp. L.lactis, L.cremoris, and the relation between S.lactis, S.lactis subsp. high and low acid-forming diacotilactis, L.lactis or L.cremoris in combination with L.plantarum are set respectively to 1.2: 0.8: 0.2: 1.8: 2.0. Table I Pleased teliyy.peg- to "bitterness 38 is good Satisfactory39 190 Sour T "voyT42 24 Hepa HOHtp- CNV-in W Is great 25 Wowlet about 23 ritslva Not equal- . 993 measured Hepaino 989 measured Appearance Colour Smell Mass fraction of moisture,% Solubility index, ml of crude residue The total number of viable cells of the milk-acid bacteria in 1 g of the drug, billion Number of viable cells: aroma bacteria in 1 g, billion lactic acid sticks in 1 g, billion The number of colonies of foreign microorganisms that have grown during the seeding of 1 g of the drug Bacteria of the group of intestinal sticks in 1 g of the drug Spores of spore-forming anaerobic microorganisms in 1 g of the drug Microscopic preparation Antagonistic activity (zone width, growth inhibition of the test culture), mm Drug activity: acidity, after 2 h of cultivation, 1 by time of clot formation, h Homogeneous powder Cream with a brownish shade Pure, sour-milk 4,5 A, 5 160 98.0 0.54 Not detected Not detected Not found Gram-positive cocci and sticks, single and in short chains 42 4.5 Sourdough prepared directly from the drug time of clot formation, h acidity, T Volatile fatty acids, ml of 0.1 N. NaOH solution Acetoin diacetyl (alkaline staining), used Carbon dioxide, when heating a ferment sample to 90 ° С, mm Appearance Color Smell Mass fraction of moisture,% Solubility index, ml of crude residue The total number of viable cells of lactic acid bacteria in 1 g of the drug, billion The number of viable cells in 1 g of the drug, billion: aromatic bacteria of lactic acid sticks The number of colonies of foreign microorganisms that have grown during the seeding of 1 g of the drug Bacteria of the group of intestinal sticks in 1 g of the drug Spores of spore-forming anaerobic microorganisms in 1 g of the drug 1409195 2 Continued table. 3 . 15 98 1.8 .five 15 Homogeneous powder Cream with a brownish tint Pure, sour-milk 4.7 A, 7 180 95 0.60 Not detected Not detected Not found Microscopic preparation Antagonistic activity (width of the zone of growth inhibition of the test culture), mm Drug activity acidity, after 2 h of cultivation, T at the time of formation of the cone, h Sourdough prepared directly from the drug: time of clot formation, h acidity, t Volatile fatty acids, ml 0.1 n. NaOH solution Acetoin diacetyl (alkaline staining), beats. Carbon dioxide when heating the leaven sample to 90 С, mm Continuation of table 4 Gram-positive cocci and sticks, single and in short chains 6.5 41 14 95 1.7 17 “J V H