The Hypoglycemic Activity of Lactic Acid Bacteria Isolated From Medicinal Plants of Uzbekistan and Their Probiotic Potential

The study aimed to evaluate the hypoglycemic activity of lactic acid bacteria isolated from the medicinal plants possessing antioxidant and hypoglycemic properties growing in Uzbekistan and analyze their prоbiotic properties. Four plant isolates of lactic acid bacteria (LAB) and three commercial strains were used in the study. Alimentary hyperglycemia was induced in white outbred rats, and glucose level was measured before and after treatment with lactic acid bacteria according to the experiment scheme using an intraperitoneal glucose tolerance test. The investigation was shown that strains of Lactobacillus kunkeei 1, L. рlantarum TK1, L. рlantarum KA3, Enterococcus faecium effectively reduce postprandial hyperglycemia in rats. Moreover, evaluation of their probiotic properties: sensitivity to antibiotics, simulated gastric juice, simulated juice of small intestine, bile, and elevated concentration of sodium chloride, demonstrated that the strains Enterococcus faecium 1, Lactobacillus kunkeei 1, L. рlantarum TK1, L. рlantarum KA3 meet the criteria for probiotics. These strains could be considered promising candidates for the preparation of probiotic preparations intended not only to correct gut microbiota but also to maintain normal blood glucose levels. Review Article Amirsaidova et al.; JPRI, 33(60B): 445-457, 2021; Article no.JPRI.79979 446


INTRODUCTION
It is known that diabetes is a common metabolic disease, occurs when the pancreas does not produce enough insulin (the hormone that regulates blood glucose) or when the body does not respond to the insulin produced [1]. In the last decades, the incidence of diabetes has been steadily increasing, and it was 451 million patients in 2017 [2]. Five million people died from diabetes this year [3].
Diabetes can damage blood vessels, eyes, kidneys, and nerves, significantly increase the risk of heart disease and stroke [4], are a significant economic and social problem. Type 2 Diabetes Mellitus (T2DM) is characterized by an increase in fasting blood glucose (FBG) and glycosylated hemoglobin (HbA1c), which indicates impaired glucose metabolism [5]. Although there are many antidiabetic drugs, the therapies for this pathology are not perfect. Most people with diabetes follow a lifestyle and diet plan to improve the effectiveness of their treatment, and for the most part, they prefer using natural medicines and traditional therapies. There is a large amount of evidence that the intestinal microflora composition is associated with the development of T2DM [6]. A close interaction was shown between T2DM and compositional changes in the gastrointestinal tract (GIT) microbiota, with a relative decrease in the number of Firmicutes and an increased concentration of Bacteroidetes and Proteobacteria in patients with T2DM [7,8]. Recent studies demonstrate that some strains of lactic acid bacteria of the species Lactobacillus rhamnosus, L. plantarum, L. gasseri, along with probiotic properties, have both the ability to lower blood glucose levels and antioxidant properties and have great potential for the treatment of type 2 diabetes [9,10,11].
According to WHO, in Uzbekistan, 8.7% of the population suffers from diabetes mellitus, and 2% of the total number of deaths is caused by diabetes mellitus [12]. Probiotic preparations from local microorganisms on the Uzbek market have not been previously studied, hypoglycemic agents.
Taking into consideration that microorganisms isolated from the geographical area in which they will be used, have a competitive advantage over other "foreign" representatives [13], the study of the ability of local strains of bacteria to reduce blood glucose levels will allow the selection of effective strains which, along with the probiotic properties will possess the ability to maintain normal blood glucose levels.
The aim of this research is to study the hypoglycemic activity and probiotic properties of lactic acid bacteria isolated from local flora and commercial preparations.

Microorganisms
The Lactobacillus plantarum TK1 strain isolated from Jerusalem artichoke root; the strain of Lactobacillus plantarum MAL, isolated from flowers of low mallow (Málva neglecta); Lactobacillus kunkeei 1, isolated from dandelion flowers (Taraxacum officinale); Lactobacillus plantarum KA3, isolated from the leaves of the Ayuga Turkestanica; Lactobacillus rhamnosus D, Enterococcus durans 1 and Enterococcus faecium 1, containing in the commercial preparations Lactobacterin and Bifidumbacterin PL have been used in the study. Plants used for the isolation of lactic acid bacteria were selected based on known antioxidant and hypoglycemic properties. De Mann, Rogosa, Sharp (MRS) medium (HiMedia) was used for isolation, purification, and growth of lactobacilli. All strains were stored in the freeze stock of the Laboratory. The dried culture was restored by double culture in MRS broth to study the microbial properties, and a suspension containing 109 CFU / ml was used [13].

Determination of Hypoglycemic Activity
Healthy 115 white outbred rats (both sexes) weighing 150-180 g were used for the experiments. They were quarantined for at least 10-14 days [14,15] and divided into 23 groups, each containing five animals ( Table 1). The study of the hypoglycemic activity of the samples (preparations) was carried out using an intraperitoneal glucose tolerance test [16]. Briefly, 30 minutes after the administration of the tested bacterial suspension to the groups No 1-23, the animals of all groups (except for the intact one) were injected with glucose in the form of an 8% solution, at a dose of 2 g / kg (5 ml / 200 g). Fifteen minutes after the glucose injection in all animals in ether anesthesia (ether was administered by inhalation), blood was taken from the cardiac region. The criterion for assessing the pharmacological activity was the normalization of blood glucose levels.
The blood was placed in a serological tube without anticoagulant and centrifuged at 3000 rpm for 10 minutes for the glucose concentration measure. Next, the concentration of glucose in the obtained serum was determined on a biochemical analyzer "HUMALYZERPrimus" (semi-automatic), manufactured by "Human GmbH" (Germany), with metrological characteristics: 340, 405, 500, 546, 620 nm, reagent consumption 400 μl.

Determination of antagonistic activity
The antagonistic properties of the studied cultures were determined by the method of spots on agar [17]. Briefly, the dots of test strains were grown on the surface of the MRS agar and covered with the second layer of the soft agar containing 10 7 CFU/ml of the indicator strain. The plates were incubated for 24

Determination of bile and different concentrations of NaCl tolerance in the environment
The bile tolerance of bacteria, their ability to grow at increased concentrations of sodium chloride were determined according to the Guidelines 4.2.2602-10. [18]. Briefly, 1 ml of 109 CFU/ml suspension of the tested culture was added to the growth medium containing 0,2; 0,3; 0,4 and 0,6% of ox bile (HiMedia) and 2; 4; 6,5% of NaCl. After incubation at 37oC for 24 hours, the number of viable cells was measured by serial dilution assay.

Study of resistance to gastric juice and small intestine juice
Survival in the presence of simulated gastric juice and simulated small intestine juice was studied according to the method described by B.M. Corcoran et al. [19]. The simulated gastric juice with pH=2 and simulated intestinal juice with pH=8 were prepared according to Cunha et al. [20]. 1 ml of test culture suspension containing 109 CFU/ml was added to the 9 ml of simulated juice, and viable cells number was determined in 30; 60; 90 min for gastric juice and in 1; 2 and 3 hours for intestinal juice by serial dilution assay.

Study of Physiological and Biochemical Properties of Strains
The test for catalase activity for the production of lecithinase, hemolysin, gelatinase, and amylase was carried out according to Guidelines for preclinical study for selection, verification, and storage of industrial strains used in the production of probiotics [17].

Evaluation of the Hypoglycemic Activity of Microorganisms in the Model of Alimentary Hyperglycemia in Rats
As a result of intraperitoneal administration of glucose in animals, a significant increase in glucose level in the blood serum was observed, which was a sign of hyperglycemia. However, the preventive intake of different strains of lactobacilli leads to a decrease in glucose levels ( Table 2).

Probiotic Properties
Probiotic properties ensure the survival of microorganisms in the gastrointestinal tract and their beneficial effect on the host organism. For the probiotic properties characterization, isolates with the most pronounced hypoglycemic properties were selected, and their antagonistic activity, resistance to bile, gastric juice, small intestinal juice, and NaCl were studied.

Fig. 5. Survival of isolates in the presence of different concentrations of NaCl
All the studied strains proved to be resistant to both simulated gastric juice and small intestine juice, ensuring their survival under stressful conditions during the gastric passage.

Resistance of strains to various concentrations of NaCl
The results showed that all cultures are resistant to 2%, 4%, and 6.5% NaCl in the medium, with an increase in salt concentration to 6.5%, the cells number did not fall below 10 9 . In the case of Enterococcus faecium 1, Lactobacillus kunkeei 1, and Lactobacillus plantarum KA3, the number of viable cells at a salt concentration of 6.5% did not differ from the initial one; in Lactobacillus plantarum TK1, it decreased by 1 log.
All studied cultures showed resistance to the presence of 6.5% salt in the medium, which indicates their stability during the production process.

Physiological and Biochemical Properties of Strains
In the cultivation process, microorganisms secrete various proteolytic enzymes into the external environment, which can be divided conditionally into two groups: the first group should include enzymes that take part in the metabolism of microorganisms (respiration, nutrition). They break down carbohydrates, proteins, peptides, amino acids, resulting in the formation of food and metabolic products easily digestible by microorganisms -acids, peroxides, indole, hydrogen sulfide, etc. The second group should include enzymes related to pathogenic factors (hyaluronidase, fibrinolysin, plasma coagulase, hemolysin, lecithinase C, lysozyme) neuraminidase) [18]. To determine the absence of synthesis of enzymes that are virulence factors, we studied catalase, lecithinase, and hemolytic activity.

Enzymes Involved in Metabolism
Gelatinase production test. The investigated strains do not thin the gelatinous medium.
Amylase production test. Amylase production is judged by forming transparent hydrolysis zones around crops in potato agar. The results showed that the cultures of Enterococcus faecium 1, Lactobacillus kunkeei 1 produce amylase. Lactobacillus plantarum KA3 and Lactobacillus plantarum TK1 did not give clear zones of starch hydrolysis around the inoculation on potato agar.
All studied cultures showed the absence of pathogenic factors.

DISCUSSION
Diabetes is a metabolic disease characterized by hyperglycemia, which strongly affects both the patient's health as well as the socio-economic development of the country. As of today, there are no cures for diabetes. The high cost and side effects of hypoglycemic drugs and the increasing number of diabetics have led to finding alternative, natural treatments. The mechanisms of action of the drugs currently used are as follows: a decrease in the flow of glucose into the blood (α-glucosidase inhibitors and biguanide); an increase in the amount of insulin (insulin and sulfonylurea injections), and an increase in insulin sensitivity (glucagon-like peptide-1, GLP-1) [21].
It is also proved that potential beneficial microbes, when isolated from the local source, would be more resistant to particular spices and herbs and have an advantage over other currently available probiotics in terms of stability, viability, and ultimately functionality after consumption [25]. In this regard, we have devoted work to determining the hypoglycemic effect of 7 strains isolated from local sources to study their probiotic potential. Some studied strains are already used in the composition of probiotic preparations produced in Uzbekistan (Enterococcus durans, Enterococcus faecium), others were isolated and studied by us in the framework of early experiments -Lactobacillus plantarum mal [26], and still others were isolated for the first time from medicinal plants possessing antioxidant and hypoglycemic properties (Lactobacillus kunkeei, L. plantarum TK1, L. plantarum KA3, Lactobacillus rhamnosus D). The ability of these microorganisms to lower blood glucose levels has been studied for the first time.
Our experiment studied the effect of a single dose of live bacteria cells on postprandial hyperglycemia in a model of alimentary hyperglycemia in rats. As a result, it was found that strains Lactobacillus kunkeei 1, L. plantarum TK1, L. plantarum KA3, Enterococcus faecium 1 have a robust hypoglycemic effect; when administered to rats, a decrease in blood glucose levels to the level of intact rats (100%) was observed. A moderate effect was observed with the introduction of Lactobacillus rhmanosus D (58.57%) and relatively low -in Lactobacillus plantarum MAL and Enterococcus durans (43.31% and 37.13%, respectively). Most lactic acid bacteria use glucose as a food source. Therefore, we assume that the studied lactic acid bacteria reduce postprandial blood glucose by suppressing glucose adsorption due to its utilization. This assumption is also confirmed by the results obtained by Tabuchi et al. [27].
The degree of glucose reduction varied slightly depending on the dose; however, a significant difference was observed between the effects of different strains. That indicates that not all lactic acid bacteria exhibit the antidiabetic effect, which is strain-dependent. The same conclusion was made when the different effects on blood glucose were found in L. Rhamnosus, and L. Bulgaricus strains in the work of Honda et al. [28].
Our study used the L. kunkeei strain, which had not previously been isolated in Uzbekistan. The isolate is isolated from the medicinal plant Taraxacum officinale, which has medicinal properties such as antioxidant activity, lowering cholesterol, and regulating blood sugar levels. L. kunkeei was firstly isolated from fermented wine and identified as a new species based on the 16SrRNA gene sequence in 1998 [29], and later the species was characterized as fructophilic lactic acid bacteria [29]. The properties of representatives of the species L. kunkeei are poorly studied, antidiabetic properties are shown in this study for the first time, and it is not previously used in commercial preparations.
The aim of screening the hypoglycemic properties of lactic acid bacteria was to elaborate the probiotic preparations to help keep the average glucose level in the blood. Although lactic acid bacteria have a GRAS (generally recognized as safe) status, not all could be considered probiotics. In order to claim that a bacterial strain is a potential probiotic, the FAO/WHO [30] has established guidelines with safety and functional criteria. They include antagonistic activity and strain behavior under conditions that mimic the GIT. That allows a selection of strains likely to survive such conditions, further investigating their potential as probiotic cultures.
We analyzed these probiotic properties of the isolates, which demonstrated hypoglycemic activity. Strains showed good adaptation to bile, intestinal juice at pH=8 and NaCl, and moderate to low gastric juice tolerance. E. faecium and L. kunkeei were able better survive simulated stomach juice (2x10 5 and 2x10 6 in 1 hour) rather than L. plantarum TK1 and L. plantarum KA3 (4x10 4 and 2x10 4 in 30 min). A strain-dependent tolerance to conditions similar to those found in the GIT was also observed in L. plantarum strains isolated from Bulgarian cheeses [31] and Fiore Sardo cheese [32].
Safety assessment must also include the lack of harmful activities, such as catalase, gelatinase, and hemolysin activity. Hemolysis is a common virulence factor among pathogens, facilitating iron availability to the microorganism and causing anemia and edema in the host [33]. Iron is a micronutrient that acts as a cofactor for several enzymes and is thus required for the growth of these microorganisms [34]. Lactobacilli can grow without iron, giving them an advantage in the natural environment, in competition with pathogenic bacteria [35]. However, some studies [36,37] have shown lactobacilli strains with hemolytic activity. The hemolytic activity was not detected in the four tested strains in our study.
These results agree with those found by Cunha et al. [20], who also detected no hemolytic activity in 30 Lactobacilli strains isolated from the stools of a Brazilian newborn infant. Similarly, Krõll et al.46, did not detect hemolytic activity in 93 lactobacilli strains isolated from Estonian and Swedish children (1-2 y old).
Lactobacilli produce metabolites such as organic acids, fatty acids, hydrogen peroxide, and bacteriocins, which can inhibit the growth of pathogenic bacteria.65 In our study, four lactobacilli were assayed for antimicrobial activity against ten pathogenic bacteria. All of them exhibited strong antagonistic activity. The nature of this activity will be investigated in our further researches. These results are in agreement with Cunha et al. [20], Martin et al. [38][39][40][41][42], Maragkoudakis et al. [36] that also detected inhibitory activity of lactobacilli isolates.
Considering the compliance of the isolate with the criteria of probiotic microorganisms (survival in SGS, the absence of pathogenic enzymes, and high antimicrobial activity (Tables 1, 3), it will be further investigated in the author's laboratories to evaluate their other probiotic safety characteristics to be considered as a new probiotic strain with a set of valuable properties

CONCLUSION
According to the result of the research work, Lactobacillus kunkeei, L. Plantarum TK1, L. Plantarum KA3, Enterococcus faecium reduce postprandial hyperglycemia in rats, and the antidiabetic effect depends on the bacterial strain.
The strains Enterococcus faecium 1, Lactobacillus kunkeei1, L. Plantarum TK1, L. Plantarum KA3, meet the criteria for probiotics and can be used to prepare probiotic preparations.
The hypoglycemic properties of Lactobacillus kunkeei1, Enterococcus faecium1, L. plantarum TK1, L. plantarum KA3, together with their probiotic properties and the absence of virulent enzymes, makes it possible to consider them as promising candidates for the preparation of probiotic preparations intended not only to correct gut microbiota but also to maintain normal blood glucose levels.

DISCLAIMER
The products used for this research are commonly and predominantly use products in our area of research and country. There is absolutely no conflict of interest between the authors and producers of the products because we do not intend to use these products as an avenue for any litigation but for the advancement of knowledge. Also, the research was not funded by the producing company rather it was funded by personal efforts of the authors.

CONSENT
It is not applicable.

ETHICAL APPROVAL
It is not applicable.