RU2239184C2 - Method for detecting bactericidal activity of milk - Google Patents

Abstract

FIELD: biochemistry.

SUBSTANCE: the present innovation deals with milk biochemistry and methods to evaluate the capacity of milk biologically active substances to inhibit the growth of microflora, Escherichia coli, in particular. The method deals with evaluating the growth of E. coli test culture upon beef-extract broth (BEB) supplemented with whey according to altered values of optic density. One should prepare milk whey out of milk sample of 50 cu. cm volume by adding 10%-sulfuric acid solution till obtaining a casein clot to be further removed due to filtration followed by restoration of medium reaction up to pH = 6 by multi-purpose indicator. One should add whey and E. coli test culture at optical density of 0.45-0.55 by PEC at red-color light filter (wave length being 550 nm) into BEB. One should evaluate bactericidal activity of milk by BEB's optical density by adding E. coli test culture and tested whey as compared with BEB's optical density and test culture without addition of tested whey at thermostating for 24 h at 38 ± 1 C followed by calculations according to certain formulas. The innovation enables to objectively evaluate milk capacity to inhibit the growth of E. coli.

EFFECT: higher efficiency of detection.

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Description

The invention relates to the field of biochemistry, in particular to the biochemistry of milk. This method is necessary to assess the biochemical, sanitary and hygienic and technological properties of milk. The method allows you to evaluate the ability of biologically active substances of milk to restrain the growth of microflora, in particular Escherichia coli. Evaluation of the bactericidal activity of milk is of great practical importance, since milk is a dietary food product. Milk with high bactericidal activity inhibits the growth of Escherichia coli and hay bacillus, putrefactive microflora, which increases the quality and dietary properties, prolongs the shelf life of milk. Studies in this area suggest that bactericidal activity can serve as a factor in assessing the genotype of an animal, since the chemical composition of milk is due to breed characteristics

Known methods for determining the bactericidal activity of milk, such as a method for determining the number of mesophilic aerobic and facultative anaerobic microorganisms (KMAFAnM) [1] or the total microbial number, a biocalorimetric method for determining the total number of bacteria [2], as well as a potentiometric method for determining the duration of the bactericidal phase [3 ] milk is based on the measurement of bacterial contamination of milk.

The essence of the bio-calorimetric method is the calorimetric measurement of the heat power released during the life of microflora, which characterizes the physiological activity of microflora. The determination is carried out in the test milk in comparison with the pasteurized sample of the same milk on the MKM-C microcalorimeter. The disadvantages of the method are that it characterizes to a greater extent not the ability of milk to restrain the growth of microflora, but its bacterial contamination, as well as the need to use a microcalorimeter, which is rarely found in laboratories.

The potentiometric method for determining the duration of the bactericidal phase is based on measuring the redox potential (ORP) of milk, which decreases with intensive bacterial growth. The analysis is carried out on a potentiometer KSP-4. The disadvantages of this method are the indirect determination of bactericidal activity by the amount of microflora present, and the use of special equipment.

The closest and most common way to determine the bactericidal activity of milk is the method for determining KMAFAnM, based on the ability of mesophilic aerobic and optionally anaerobic microorganisms to reproduce on solid nutrient agar at (30 + 1 ° C) for 72 hours. The determination is carried out by seeding dilutions of the test milk on agar at time intervals and counting microbial colonies grown in 72 hours. The end of the bactericidal phase is determined by the beginning of the intensive growth of bacteria. However, this method has several significant disadvantages. It is laborious and inaccurate, since the final result largely depends on the initial bacterial contamination of the milk.

Thus, none of the existing methods can directly assess the ability of milk to inhibit the growth of bacteria.

The aim of the invention is to develop a method for determining the bactericidal activity of milk, which allows to objectively assess the ability of milk to inhibit the growth of E. coli.

This goal is achieved by assessing the growth of the test culture of Escherichia coli on meat and peptone broth (MPB) with the investigated whey introduced into it by the change in optical density. To do this, milk serum is prepared from a milk sample with a volume of 50 cm ³ by adding a 10% solution of sulfuric acid to form a casein clot with removal of casein by filtration, followed by restoring the reaction of the medium to a pH of 6, according to a universal indicator, whey is added to the MPB and test culture of E. coli with an optical density of 0.45-0.55, measured at the FEK with a red light filter (wavelength 550 nm), the bactericidal activity of milk is assessed by the optical density of the BCH with the addition of an E. coli test culture and of the studied whey in comparison with the optical density of the MPB and the test culture without the addition of the studied serum when incubated for 24 hours at a temperature of 38 ± 1 ° C, with further calculations by the formulas.

Comparative analysis with the prototype allows us to conclude that the inventive method for determining the bactericidal activity of milk allows you to evaluate the bactericidal activity directly, and not indirectly, that is, meets the criterion of "novelty."

Features that distinguish the proposed technical solution from the prototype:

1. obtaining whey by adding a 10% solution of sulfuric acid to pH 3-4 to form a clot of casein;

2. removal of casein clot by filtration;

3. restoration of the reaction of serum medium 1 N. caustic soda solution to a pH of 6, according to the universal indicator;

4. preparation of a test culture of E. coli from a daily culture of Escherichia coli grown on agar by washing with saline and adjusting the concentration of bacteria to 0.45-0.55 FEC at a wavelength of 550 ± 21 nm;

5. the introduction of a test culture and whey in sterile BCH (volume 4.5 ml);

6. measuring the optical density of the MPB at the beginning of the experiment, after 3, 6, 9, 12 and 24 hours of temperature control at a temperature of 38 ± 1 ° C;

7. Evaluation of the results on the change in the optical density of the MPB with the addition of the E. coli test culture and the test whey in comparison with the optical density of the MPB and the test culture without adding the test serum in each time interval is carried out according to the calculation formulas 1, 2, 3.

Calculation formulas

where NM is the increase in the mass of E. coli, as a percentage of the initial value;

D ₁ - the initial optical density of the sample;

D ₂ - the optical density of the sample after a calculated period of time.

where BA is the bactericidal activity of milk after a calculated period of time,%;

NMcontrol — increase in the mass of E. coli in the control sample,%;

NMop - increase in the mass of E. coli in the experimental sample,%.

where BAsp is the average bactericidal activity of milk within 24 hours,%;

BA ₃ - bactericidal activity of milk after 3 hours,%;

BA ₆ - bactericidal activity of milk after 6 hours,%;

BA ₉ - bactericidal activity of milk after 9 hours,%;

EA ₁₂ - bactericidal activity of milk after 12 hours,%;

BA ₂₄ - bactericidal activity of milk after 24 hours,%.

Analysis of the known technical solutions in the field of biochemistry and microbiology of milk allows us to conclude that there are no signs in them that are similar to the essential distinguishing features in the inventive method, such as a direct assessment of the bactericidal activity of whey with the introduction of the E. coli test culture on a photoelectrocolorimeter, and recognize the claimed method corresponding to the criterion of "significant differences".

Execution example.

The method is based on measuring the optical density of meat and peptone broth (MPB) with the growth of E. coli in it with and without the addition of serum of the test milk.

When testing the method, two samples of unpasteurized milk obtained from different animals were analyzed. Designated them No. 1 and No. 2, and the studied parallels 1.1-1.5 and 2.1-2.5, respectively.

From each milk sample, whey was obtained by adding a 10% solution of sulfuric acid to pH 3-4 according to a universal indicator in order to remove protein - casein, which does not affect bactericidal activity, but does not allow milk to be examined by colorimetry. A clot of casein was removed by filtration, then the reaction medium (pH) was restored 1 N. caustic soda solution to pH 6, which is necessary for the normal functioning of the enzymes contained in milk serum and having a bactericidal effect.

A test culture of E. coli was prepared using a daily culture of E. coli grown on agar. The daily culture was washed off with sterile physiological saline and the bacterial concentration was adjusted to a density of 0.45-0.55 on the lower FEK scale at a wavelength of 550 ± 21 nm. The test culture was placed in sterile MPB / volume 4.5 ml / and the test whey was added. At the same time, a control sample was prepared without the addition of whey.

The optical density was measured at the beginning of the study and after 3, 6, 9, 12 and 24 hours of temperature control at a temperature of 38 ° C (table 1).

Evaluation of the results in each of the time intervals was carried out according to the formulas 1, 2 and 3 (tables 2 and 3).

Thus, whey inhibits the growth of Escherichia coli within 24 hours. The maximum bactericidal activity in both samples appears after 9-12 hours of incubation and is 83 and 96%. The bactericidal properties of milk appear after three hours of temperature control.

Using the proposed method for determining bactericidal activity provides the following advantages compared to existing methods.

1. The proposed method for the first time allows you to quantify the bactericidal activity of milk by the ability of whey to inhibit the growth of E. coli on a nutrient medium, while existing analogues evaluate it only indirectly by bacterial contamination of milk.

2. The objectivity and accuracy of the evaluation are ensured through the use of the FEC available in all laboratories.

3. The implementation of the methodology does not require expensive equipment and reagents.

4. If necessary, the evaluation of the bactericidal properties of milk can be carried out by the accelerated method for 3 hours.

Sources of information

1. GOST 9225-84 Milk and dairy products. Methods of microbiological analysis.

2. GOST 27930-88 Milk and dairy products. Biocalorimetric method for determining the total number of bacteria.

3. Peinovich V., Pigeon V., Ponamarev A. Bactericidal properties of milk when stored in frozen form. - Dairy and beef cattle breeding, 1988, No. 1, p.24-25.

Claims (1)

A method for determining the bactericidal activity of milk, comprising introducing the test sample into the nutrient medium, thermostating and evaluating the bactericidal activity of milk, characterized in that in the nutrient medium, which is used as meat and peptone broth, along with the test sample of whey, test culture E. coli with the optical density of 0.45-0.55, determined by the photoelectrocalorimetric method at a wavelength of 550 nm, the bactericidal activity is assessed by comparing the optical density of the meat the peptone broth with the addition of E. coli test culture and whey and without the latter, followed by thermostating at a temperature of (38 ± 1) ° С for 24 hours, while the results are evaluated according to the formulas below:

where NM is the increase in the mass of E. coli, as a percentage of the initial value; D ₁ - the initial optical density of the sample; D ₂ - the optical density of the sample after a calculated period of time;

where BA is the bactericidal activity of milk after a calculated period of time,%; NM _counter - increase in the mass of E. coli in the control sample,%; NM _op - increase in the mass of E. coli in the experimental sample,%;

where BA _avg is the average bactericidal activity of milk within 24 hours,%; BA ₃ - bactericidal activity of milk after 3 hours,%; BA ₆ - bactericidal activity of milk after 6 hours,%; BA ₉ - bactericidal activity of milk after 9 hours,%; BA ₁₂ - bactericidal activity of milk after 12 hours,%; BA ₂₄ - bactericidal activity of milk after 24 hours,%.