3 experimental part

Determining the shelf life of soft cheese

The shelf life of the product is determined in accordance with SanPiN 2.3.2.1324-03 "Hygienic requirements for shelf life and storage conditions of food products" and is 5 days at storage temperature (0-6)° C and relative humidity (80 ± 2) %. In accordance with MUK 4.2.1847-defined reserve 04factor as a consequence1,5, of which the study of the main quality and safety indicators were conducted for 8 days. The safety indicators of soft cheese were controlled, which are presented in the table (32Appendix 5).
As a result of the research conducted for soft cheese without maturation from a mixture of cow's and goat's milk can be attributed to the group of perishable products. As a result, a shelf life of 5 days from the date of production is established, taking into account the time it stays in the trade network at a temperature of (4±2) ° C and relative humidity of 80-85%.
Table32 - Sanitary and epidemiological assessment of soft cheese samples from a mixture of raw materials

Name of indicator		Norma		Timing test results
				2 sut		5 sut		8 sut
				obrazeciz milk mixtures	sample from chickpea flour	obrazeciz milk mixtures	sample from chickpea flour	obrazeciz milk mixtures	sample chickpea flour	с
BGRP (coliforms) of the product weight, g	в	in 0.001-0.01 product not allowed		not found
Pathogens, Including Salmonella		wg 25 product is allowed	not	not found
Pathogens, including L. monocytogenes, in product weight, g		wg 125 product is allowed	not	not found
Yeast, CFU/g		not The number of yeast spoilage pathogens may not increase by more than 2 times the initial level detected		1,2 10^2	1,9 10^2	1,4 10^2	2,2 10^2	1,8 10^2	2,7 10^2
Moulds, CFU/g		not The number of yeast spoilage pathogens may not increase by more than 2 times the initial level detected		2,0 10^1	3,0 10^1	2,5 10^1	3,3 10^1	3,2 10^1	4,0 10^1
Bacteria of the genus Proteus in of the product weight, g		not regulated		in g1,0 of product not detected
Staphylococcus aureus in of the product weight, g		in 0.001-0.01 product not allowed		not found

5. Determination of allergenicity indicators for soft cheese

The allergenic properties of the cheeses were evaluated in a "delayed" hypersensitivity test on laboratory mice.
The study was performed in accordance with the rules of laboratory practice in the Russian Federation (Order of the Russian Ministry of Health No. 199n "On Approval of the Rules of Good Laboratory Practice" dated 1 April 2016) and the Methodological Recommendations of the FSBI "Scientific Centre for Expertise of Medicinal Products" of the Russian Ministry of Health and Social Development [110].
The research organisation followed the requirements of this approved research protocol and the laboratory's standard operating procedures (SOPs).
When handling laboratory animals, the rules for humane treatment were observed. Animals were handled by appropriately qualified and trained personnel. This study was conducted in accordance with internationally recognised standards (Animal Welfare Act, Guide for Care and Use of Laboratory Animals).
The species of experimental animals were chosen according to the methodological guidelines for assessing the allergenic properties of pharmacological substances described in "Guidelines for preclinical studies of medicinal products" [110]. [110].
The number of animals in the group is sufficient for a complete, reliable record of the parameters studied.
Accommodation of animals was carried out in accordance with the SOP "Animal Intake and Quarantine". Cages with animals were placed in a quarantine room. The duration of quarantine for all animal species was 14 days. During the quarantine period, each animal was weighed and physically examined at least once 2(day and1 day 14). Throughout the study, all animals were inspected once a day in their cages to detect sick or fallen animals.
The investigated cheeses were administered orally and subplanterally to mice. The frequency of administration and doses depended on the specific test. The administration volume was calculated individually for each animal based on the body weight value obtained after the last weighing.
Clinical observations:

• 
  Daily inspection of the animals to assess their general condition (mobility, mucous membranes, eyes and skin);
  
• 
  inspection of the injection site - one day after injection; if there are deviations from the norm - daily (assessment of the skin condition, signs of inflammation) until normalisation;
  
• 
  Daily observations to detect signs of allergic response to the study medication (signs of anxiety, assessment of skin and mucous membranes).
  
• 
  observation during animal testing for allergenic properties (assessment of reactions at the sites where authorising doses are administered).
  

Feed and water consumption of the experimental animals was assessed for each group visually, daily. The experimental animals were deprived of food 12 hours before the planned euthanasia according to the experiment scheme. Access to water was not restricted.
Experimental laboratory mice of the C57BL/6 line, irrespective of their group identity, were sensitized once, intradermally to the base of the tail with μl60 of emulsion of the test substance in a 1:1 ratio of Freund's adjuvant (PAF). The emulsion is prepared using Hanks' solution with pH To 7,5.detect sensitization after 245 hours, mice were injected with μl40 of the test substance solution in Hanks' solution into the hind paw pad (Figure 17).



Figure17 - Solution administration process
Control animals were sensitised by emulsion of PAF with Hanks' solution using the same scheme as in the experiment.
After 6-22-24 hours of testing, the swelling was measured using an engineering micrometer. The difference in thickness of both legs characterised the magnitude of the oedema, which was used to judge the intensity of the GZT response (Figure 18).

Figure18 - Measuring the size of the swelling
Delayed hypersensitivity reaction index (DI) was determined
_ГЗТ) by the difference in thickness between the experimental and control limbs.
Experimental laboratory mice of the C57BL/6 line were sensitized irrespective of their group identity by a single intradermal injection of 60 μl of emulsion of tested soft cheeses in complete Freund's adjuvant (PAF) in a 1:1 ratio at the base of the tail. A permissive dose (40 µl in Hanks' solution) was administered to mice in the hind paw pad after one day5. Control animals were sensitized by emulsion of PAF with Hanks' solution according to the same scheme as in
experience. The "delayed type" hypersensitivity reaction was assessed after 6, 22 and 24 hours by the amount of oedema. The results of the experiment are shown in the table below 33.
Table 33 - Effect of the GLF thiadiazole tested on the course of the delayed-type hypersensitivity reaction in mice

№ gr.	Sample	GZT response index
		Hours6 later	After an hour24
1	Control substance	10,396,48	11,663,32
2	Cow's soft cheese milk	48,296,58	34,027,26
3	Soft goat's milk cheese	41,725,57	31,692,83
4	Soft cheese made from a mixture of milk	43,226,46	32,443,24

Analysis of the data in Table 33 shows that the cheeses tested induce an increase in the GGT response index both 6 hours after the administration of the permissive dose and one24 hour later. However, no statistically significant intergroup differences were found. Therefore, we can talk about the individual sensitivity of mice to cheeses: in each group, a significant GGT response was observed in 3-5 mice from 10.