Probiotics in Processed Dairy Products and Their Role in Gut Microbiota Health

Abstract

Probiotics are the beneficial microorganisms, catalase negative which restore microbial balance inside the gut of humans as well as animals. Lactobacillus the earliest probiotic that have the beneficial impact on health. These “Good Microorganisms” can be obtained not only from various non-dairy products but also from processed dairy products like. Another economically viable method is microencapsulation for preserving probiotics and the stability is improved by glucose. Even the vitamins manufacturer the probiotic bacterial agents. The health benefits of probiotics include increased immunological responses, relief of lactose intolerance symptoms, therapy for diarrhea, reduction in serum of cholesterol, production of vitamin, anticarcinogenic. Probiotics play a wide range in the host body (e.g., decreasing illnesses and stress, enhancing immunity, modulation of gut microbiota, nutritional assistance, improving quality of water, etc.). So, the positive effects of probiotics help to boost animal feed value and growth and improve aquaculture breeding and hatching rates. Probiotics can lower the prevalence and severity of illnesses, showing their promise to cure or prevent COVID-19. Lactobacillus casei also interact with epithelial cells with Toll-like receptors (TLRs) to improve the production of cytokines that are important in the enhancement of cell productivity and prevent apoptosis during restoration, which promote survival and proliferation. The preservation of the human GI or lung microbiota might help prevent COVID-19, as dysbiosis plays an essential role in people’s vulnerability to infectious illnesses. Most of the experimental studies proved that bacteria isolated from processed dairy products belonged to lactic acid bacteria and are declared as probiotic bacteria. In present review, various research studies regarding significance of probiotics as well as their extraction from processed dairy products are discussed.

1. Introduction

“Probiotic” is a Greek term which meaning “for life” [1]. Over the years, there have been numerous meanings of the term “probiotic.” Probiotic means living microbiota cultures that enhance the qualities of the indigenous microbiota when supplied to people or animals. These bacteria are extensively spread in nature and are suitable for usage in food industries. Some foods are considered a good source of probiotics, including milk products, e.g., yogurt. Milk and milk products are often linked with microorganisms that replenish the digestive tract with helpful maintenance [2]. Different generations of lactic acid bacteria like Enterococcus species, Lactococcus species, different strains of Lactococcus and Streptococcus are also present in processed products of milk.

Lactic acid bacteria are a type of gram-positive bacteria that cannot produces spores and catalase negative, so they characterized in the absence of cytochrome system. Different sources of food like yogurt, milk, cheese that are dairy in nature are the good source of probiotics [2]. Most processed milk products contain the Lactobacillus, and other strains of it.

Increased taste of food and better shelf life are among the most evident benefits of LAB fermentation. The fact that LABs can produce the bacteriocins and provide health benefits, including controlling intestinal infections, improving lactose uses, reducing the ammonia level of the blood, and providing effective resistance to gastric acid and bile, makes it generally considered safe for bacteria to be used. Impacts the immunological system and decreases serum cholesterol levels. Probiotics are live, health-effecting micro-organisms when eaten. Different LAB strains, particularly Lactobacilli and Bifidobacterium which reside in the adult bacteria in the gut of humans with good therapeutic functions have been used increasingly as probiotics.

1.1 History

In 1908, Metchnikoff gave the first probiotic definition, which suggested that use of fermented dairy produce extended the life of the product. In 1956 Lilly and Stillwell decided that some growth stimulators for another microbe were secreted by a microbe. The usage of the word probiotic may lead to this beneficial impact on such micro-organisms.

Parker [1] coined the word probiotic to define the chemicals and organisms that produce microbial balance in the gastrointestinal system. This meaning has given rise to significance meaning, including antibiotics, of the word substances. Fuller refined the description of Parker and described probiotics as a living micro-organism, which has a beneficial effect on warm-blooded animal health through the restoration of native gut microbiota. The definition by Fuller highlighted the survivability and beneficial effects of the probiotic on animals. In the instance of probiotics host, Haveenar and Veld described them as ‘viable micro–organics’ in 1992, which are administered to humans or animals as mono- or mixed cultures which have a favorable effect on the health of host, by enhancing the intestinal characteristics of the micro-flora. A probiotic is a live bacterium injected in milk products based on the Salminen criteria and enhance host health and diet.

Schaafsma extends the concept and according to its definition probiotics are live bacteria which, if swallowed and absorbed in a more than intrinsic basic nutritional way, have health benefits on the host. Salminen noted that the probiotic in milk products is a microbial culture. Accordingly, the food matrix is a big indicator for the microbe and food being regarded as probiotics. However, because non-dairy food includes viable probiotic products, it was not justifiable to take milk products only as a probiotic matrix. In 2001, Probiotics were seen by Schrezenmeir and by De Vrese as live micro-organisms which have independent health effects of the place of activity.

In 2001, probiotics are known as “live micro-organisms by the WHO (WORLD HEALTH ORGANNIZATION) and FAO (FOOD AND AGRICULTURE ORGANIXATION OF THE UNITED STATES) that provide the host with a health advantage by being given adequately.” Finally, in 2014, the ISAPP changed the latter definition of probiotics to somewhat and described it as ‘live micro-organisms, which provide a health benefit on a host when administering in appropriate quantities.’ This concept has been extensively adopted by the scientific community since then and is a criterion in most government agencies to evaluate medicines, food, and supplements as probiotics. The viability of a microbe and of the related product as ‘probiotic’ is one of the essential criteria in this definition.

1.2 Lactobacilli

Lactobacillus is regarded as the earliest probiotic reported. This genus is made up of LAB group gram-positive bacteria. These bacteria in rod shape include over 183 recognized species and are frequently used in diverse commercial food processes [3]. L. acidophilus is a bacterium that has a positive impact on the host. Recent investigations have investigated the finding of normal vaginal flora of certain species producing hydrogen peroxide. The beneficial products have thus been examined in urine and vaginal tract infections among women, so it helps in the treatment of it. It was also used to treat Candida infections in the mouth (Figure 1).

Figure 1.

Types of probiotic strain.

Reticence of harmful organisms like Salmonella, Shigella and different Helicobacter comprise valuable effects mediated by Lactobacilli. Lactobacilli was also related with several additional health advantages, e.g., improved immunological response and lessening of lactose intolerance. A good function was also revealed in colon cancer for Lactobacilli. Lactobacilli strengthen the immune system and cure cancer, pancreas, fever blisters, hives. In infants who are born preterm, necrotizing enters colitis (NEC). Cheese is a milk product that may be delivered in the intestine of people by probiotic bacteria. Italian, Argentinean, and Bulgarian cheese have isolated the strains of L. plantarum.

1.3 Source of probiotics

Cheese is a milk product that may be delivered in the intestine of people by probiotic bacteria. Italian, Argentinean, and Bulgarian cheese have isolated the strains of L. plantarum [4].

1.3.1 Lactic acid bacteria in ice cream

It is referred as a frozen dairy product that is produced by freezing a sterilized mixture to incorporate air to maintain homogeneity. Milk products, sugar, dextrose, water, eggs or egg products, and different non-harmful aromas are used to form a mix that is utilized to make ice cream. Its nutritional content and their energy value are generally determined by their food value [5]. Irrespective of the mix, ice creams usually constitute great sources of dietary energy and are an appropriate substratum for probiotic proliferation.

Ice cream is considered as favorable for the probiotics as it helps in survivability and metabolic activity of probiotics and its different strains shows more benefit for transferring probiotic organisms to the body [6]. Ice creams are more advantageous than other fermented dairy products. But freezing and thawing have detrimental consequences on probiotics, including disruption of metabolic activity and cell death. There have been reports of studies designed to reduce these negative consequences.

Lactobacillus delbrueckii sub specie was investigated by Leandro [7]. Bulgaricus UFV H2b20 is kept at −16°C for 40 days in three formulations of ice cream (it contains less fat, free of fat and high-fat). Although in the three formulations (P > 0.05), LABs may be integrated into ice-cream formation and different processing parameters for different consumer groups may be handled (Table 1).

Type of ice cream	Name of the probiotics added	Method of probiotic supplementation	Viability and storage conditions (CFU/g)	References
Standard ice cream	Lactobacillus casei	The probiotic strain was added after homogenization and heating of ice cream mix	Count from 3.9 × 10^9to 3.8 × 10^8 log CFU/ml 80 days of storage at −20°C	[9]
Standard ice cream	L. plantarum	The probiotic strain (encapsulated within a calcium—alginate/chitosan microcapsules containing insulin) was added after homogenization	2.3 × 10^7 after storage of 90 days at –20°C	[10]
Yog ice cream	Lactobacillus acidophilus La5	Encapsulated probiotic strain (alginate-based) was added into the ice cream mix	After 60 days of storage at –18°C, the viable probiotic count was approximately 1 × 10^7	[11]
Standard ice cream	L .acidophilus ATCC 4356	Ice cream mix was fermented with L. acidophilus prior to freezing	After 90 days of storage at –18°C the viable probiotic count was 1× 10^6	[12]

Table 1.

Use of different strains of probiotics in ice cream [8].

1.3.2 Agitated milk products

It is an old method that is used for the preservation of food by the development and activity of microorganisms. Moreover, the additional benefit of fermentation is the formation of metabolites such as bacteriocins, helps in improved nutritional status and for the sensory properties of foodstuffs, and helps in reduced toxic and anti-nutritional components [13]. Due to these positive impacts, these food stuffs are important to human diets since ancient times.

In 2001 regarded probiotics are known to be “living microorganisms by the WHO and FAO that offer health benefits to the host if properly supplied”. In 2014, the definition of probiotics changed by the department of International Scientific Association for Probiotics and Prebiotics (ISAPP) as a “lived micro-organism that, when supplied adequately, conveys a health advantage to the host.” In 2014, Yerlikaya described that, Lactobacillus and varieties of Bifidobacterium are the most frequent probiotics in fermented food items. In 2012, Mishra find out that dairy-based matrix is appropriate to proliferate probiotics by providing a high carbon and necessary amino acid source owing to the hydrolyzing of lactose and the usage of casein in the proteolytic system.

Traditional white cheese is widely referred to as Lighvan cheese in Tabriz marketplaces. It was originally prepared of raw ewe’s milk, raw goats’ milk, and raw cow’s milk and/or mixed with them from time to time. This sort of cheese is popular and often consumed across Iran and has significant economic and nutritional benefits because of its attractive organoleptic qualities. In the manufacturing and maturation of cheese, lactic acid bacteria (LAB) are involved. Many genera are involved in LAB, such as Lactobacillus, Streptococcus, Enterococcus and Leucon Stoc.

1.4 Mechanism of action of probiotic

In 2012, Bermudez studied that the principal mechanisms of probiotics action include to enhance the epithelial barriers, increases in the adherence to gut mucosa and microbial adhesion, generation of antimicrobial compounds and regulation of immune systems. This process is shown in Figure 2: that illustrates how these processes appear in the intestinal mucosa, as a schematic illustration. Lactic acid from various carbohydrates (e.g., carbon sources are produced in the micro-organisms of the LAB Group [15]. Pereira [16] conducted a study in which various antibacterial processes of probiotic activity are connected to these components.

Figure 2.

Probiotics as an alternative antimicrobial therapy: current reality and future directions [14].

1.5 Development of probiotics

As we know that during human development microbiota changes in the gut of human. The newborns’ gut is completely sterile yet colonization of several types of bacteria starts shortly after delivery. The first- and second days following delivery have been demonstrated to be present in newborn feces, including coliforms, enterococci, clostridia, and lactobacilli. Bifidobacterial start colonization in three to 4 days and prevail about the fifth day. Coliform numbers drop at the same time. In feces, 1 log count of bifidobacterial is more prevalent in infants than those fed by bottle. Bottle-fed babies show greater levels of strains of Enterobacteriaceae, and other putrefactive bacteria, indicating that babies that are given breast are resistant to gastrointestinal diseases than infants fed by bottles. To ensure a person’s diet and health, the gastrointestinal system also modify in addition to the changes in the microbiome that happen throughout human aging. Using antibiotics, for example, might disrupt the balance of gut microbiota, reduce bifidobacterial and lactobacilli count and increase clostridium. This imbalance may result in diarrhea in senior citizens and in those who are immunocompromised.

1.6 Beneficial host response

Some probiotic methods elicit many positive reactions from the host. Most of the effects of these products include: (1) exclusion and competition for pathogen-cell adhesion to epithelia, (2) inborn immune stimulation, (3) compete for nutrients and prebiotically products, (4) manufacture of antimicrobial substances and consequent pathogenic antagonism; DC: dendritic cells; LI: interleukin; M: intestinal cells M. IEC: intestinal cells M (Figure 3) [17].

Figure 3.

Probiotics latest advances [17].

1.7 The probiotics of next generation

The idea of traditional probiotic products, taken from a limited number of microorganisms, is connected to the observation of the health benefits to both humans and animals from the daily consumption of LAB-fermented food. The word ‘probiotic’ was therefore associated with bacteria that promote health [18]. The development of knowledge of human microbiota of intestine and its importance for disease and health has led to the discovery of several new bacteria that plays a significant role in the health of human through therapeutic modulation of the intestinal microbiota and are called NGP. The study is very much in the interest of investigating the probiotic potential of commensal bacteria. NGPs are defined as “living micro-organisms identified by comparative analyses which provides benefits to health of the host when properly administered”.

1.8 Probiotics in fermented milk

The largest existence of probiotic products in the dairy sector is fermented milk. Several studies have successfully applied probiotic strains to milk fermentation and have induced desirable textural properties, that is apart from inducing health-promoting effects. Highly nutritional value makes its widespread availability and the most widely utilized probiotic milk products. Many commercially manufactured probiotics fermented dairy products are commonly used throughout the world (Table 2). Gao in 2019 described that the probiotic products of Kefir and Koumiss are the natural fermented milks mostly used in many parts of the world. Many studies have indicated that probiotic strains are incorporated in traditionally fermented milks that aids to improve their positive health impacts. For example, in a natural milk product (lait curd) of Senegal, Parker et al. [19] integrated L. rhamnosus GG. B. lactic Bi-07 and L. acidophilus NCFM, Wang et al. [20] have been integrated into natural milk that aids to improve health of intestine and the immunity of host cell. Many probiotic bacteria in conjunction with traditional probiotic bacteria that are reported to be use in fermented milk to improve the flavor and other characteristics [21, 22], that was linked with functional food. In yogurt, Streptococcus Thermophilus and Lactobacillus delbrücckii sub specie bulgaricus, Lactobacillus plantarum P-8 fermentation have the capacity to improve the yogurt flavor profile by producing 3-methylbutan, acetone, onanal, 2-heptanone, hexanale, (E)-2-octenal and 2-nonanone, compared to controls [23]. In the same way, a high acetic acid, acetoin, 2-butanone, caproic acid, butyric acid, and 2-pentanone content were found in fermented milk containing L. casei DN-114001 compared to control group [24].

Dairy products
Pediococcus acidilactici SMVDUDB2	Kalarei, a fermented cheese product	80% survival rate at pH 2.0 and 3.0 and 0.3% bile salt concentration, high hydrophobicity affinity (33%) with ethyl acetate, auto aggregation (77.6%), antibacterial activity against Bacillus subtilis, Mycobacterium smegmatis, Staphylococcus aureus, Proteus vulgaris, and Escherichia coli, and EPS production (2 g/l)	[11]

Table 2.

Probiotics in the dairy industry.

1.9 Probiotics in yogurt

Yogurt is a functional ingredient that contain probiotics, so there is great interest in producing probiotic yogurts that are either fermented or incorporated into yogurts with different strains of probiotic. Several commercially produced probiotic yogurts are widely utilized worldwide. A standard yogurt is a fermented milk product traditionally made of L. delbrueckii subsp. bulgaricus and S. thermophilus by fermenting the milk. As yogurt starter cultures can survive in human GI tract [25]. These may be considered probiotic because they have health-promoting effects [26]. However, all the strains of the yogurt starter culture worldwide are not identical and, therefore, the probiotic potential of yogurt starter culture in general remains controversial.

The physiochemical, sensory, and microbial characteristics of the yogurts produced by many probiotic strains comparable to the traditionally produced yogurts are much better in many cases. Of course, certain probiotics, such as L. plantarum and L. acidophilus, can reduce the bisphenol A (estrogenic substance) content of yogurts considerably [27]. The yogurt metabolism leads to large amounts of unmetabolized lactose and residual galactose in yogurts, which have been shown to be more metabolized (full use of lactose and efficient galactose degradation) by probiotic L. plantarum WCFS11 [28].

1.10 Probiotic butter and cream

There have been several products in which probiotic products have been incorporated due to its widespread benefits, and butter is also used. This is not limited to fermented milk, yogurt, and cheese. Butter, mainly made up of fats, has many health advantages. Emergent evidence, however, suggests that many cardiovascular diseases and diabetes have a high content of saturated fatty acids in butter [29]. Some probiotic bacteria have been reported to reduce the cholesterol content of cream and butter [30] (L. casei subsp. For example, in cultivated cream creams high contents of capric, butyric and caproic acid were produced when a blend of probiotic strains, including Bifidobacterium bifidum, L. acidophilus, S. thermophilus, and L. bulgaricus, was used in the fermentation of creams enriched by 2% (each) sunflower oil, hazelnut oil and soy oil [31]. The increase of the contents of linoleic and α-linolenic acid in probiotic cream in relation to control cream was observed in another study following Bifidobacterium lactis fermentation [32]. In nondairy butters, for instance the peanut and sunflower-produced butters, cocoa and flaxseed oils, probiotics are more commonly used.

1.11 Probiotics in powdered milk and infant formulas milk powder

Probiotics aid in development of an effective immune system by changing the microflora of the intestine in infants. Probiotics and prebiotics are increasingly added to infant formulae. Probiotic dispensation of bifidobacterial and the strains of lactobacilli in neonatology has developed in worldwide. B. bifidum and L. acidophilus, dispersed into infant formula (109 CFU/250 mg tablet), have been reported to continue to be more resistant by comparing with breast milk, after storage capacity at 4°C or 6 h [33].

1.12 Technological challenges for dairy products viability

The survival of probiotics is highly crucial as it provide the highly recommended efficiency of probiotics products. During food manufacturing, storage, and gastrointestinal movement, probiotics face multiple stress situations [34]. A minimum of 106 CFU/g of B. bifidum and 107 CFU/g of L. acidophilus in fermented milk are required for several international standards. The probiotic fermented milk should contain at least 107 CFU/ml of live bifidobacteria at the time of consumption in Japan (according to its Association of fermented milk and lactic acid drinking) [35]. The incidence of oxygen in processed dairy products has an impact on most of the probiotic strains and their survival. The oxygen-induced toxicity in milk products poses a major technological obstacle to the development of probiotic fermented milk and yogurt. Bifidobacterium species have an anaerobic metabolism of an intestinal origin, which implies that they depend completely on fermentation.

1.12.1 Role of oxygen in viability of yogurt probiotics

The potential reduction of yogurt bifidobacteria after storage in different regions of the world has been documented as oxygen toxicity [36, 37]. Expenditure of probiotics to dissolved oxygen leads to a build-up intracellular of harmful oxygenic metabolites, like superoxide anion [38]. A high level of oxygen is unavoidably included in the product by several processing processes involved in the processing of milk products (e.g., agitation and mixing procedures). In addition, some packing materials during the storage period enable the transmission of ambient oxygen into the food, such as high impact polystyrene packaging—a commonly used packaging material for yogurt worldwide because of its vision, strength, and hygiene levels [39]. The use of oxygen-impermeable containers, process of two-stage fermentation, acid and bile salt resistant strains, probiotics microencapsulation and prebiotics are significant preconditions for enhancing stability and viability of yogurt-based probiotics [40].

1.12.2 Studies demonstrating the viability of probiotics by oxygen

In several investigations, oxygen scavengers or antioxidants have been found to be beneficial agents for increased probiotic viability. Ascorbic acid (Vitamin C), for example, has been observed to improve L. acidophilus’ survivability in yogurt [41]. Glucose based oxygen scavenger was evaluated for its impact on probiotic development and survival in dairy products [42]. In yogurt prior to fermentation, glucose oxidase (62.25 ppm) was increased to 69.02–86.03% and the number of Bifidobacterium longum by up to 40.32% compared to the control level [43].

1.12.3 Role of glucose for improving probiotic stability

Some probiotic bacteria have a significant pH susceptibility below 3. It is stated that glucose has been included into the growing medium for improving probiotic stability. Corcoran and others observed glucose improving L. rhamnosus GG’s survival at a pH lower than 3. The addition of GL (1 to 19.4 mM) improved L. rhamnosus GG survival in artificial gastric juice from 6.4 to 8 log10 CFU/ml [44]. Therapeutic adaptation of the acid stress was also described as ways to improve Bifidobacterium species stress tolerance and biological characteristics [45].

1.12.4 Probiotic microencapsulation from different manufacturing processes

Another economically viable method is microencapsulation for preserving the probiotics against various treatment procedures and for ensuring their distribution to the human body in a necessary amount. Different studies have indicated improved probiotic survival when embedded. In the case of oligosaccharides, gelatin, inulin and xanthan gum, alginate materials (i.e., sodium alginate and human-like collagen, gelatin-based microspheres, alginate-like gum, gum-Arabic derived from cellulose, maltodextrin, vegetable protein, pectin hydrogel beads, carrageenan, and other proteins) are also supplied with the use of probiotics intended for use in milk products [46].

1.13 Vitamins and probiotics

Vitamins are generally classed to include vitamins (A, D, E and K), fat-soluble or to be water-soluble that including vitamins C, biotin (vitamin H or B7), vitamins B—thiamin (B1) and B—thiamin (B2) and riboflavin (B3) (B12). While fat-soluble vitamins are key cell membrane components, water-soluble vitamins are used as coenzymes, usually conveying chemical groups. People are unable to synthesize most vitamins and must thus be extracted exogenously. Using vitamins can be an alternative to reinforcement using chemically synthetized pseudo-vitamins that is more natural and consumer friendly.

Probiotic bacteria empower a beneficial effect on the host immune system and on the gut microbiota composition and function. In addition, vitamin synthesis has brought various health benefits to the host. Probiotic bacteria, mostly of the Lactobacillus and Bifidobacterium genus, provide several health advantages. The vitamin K, and most aquatic-soluble B vitamins, including as biotin, cobalamins, folates, nicotinic acid, pyridoxine, riboflavin, and thiamine, can manufacture probiotic bacterial agents, members of the gut microbiota, in humans. Probiotic bacteria have been widely investigated to produce B-vitamins, notably folate and riboflavin (B2). Several LAB species manufacture these vitamins, frequently in high quantities, and are therefore often present in fermented foods (e.g., Lactococcus lactis, Lactobocillus gasseri, and Lactobacillus reuteri) and Bifidobacterium (e.g., Bifidobacterium adolescentis). In addition, higher production of vitamins has been achieved through metabolism. Folate biosynthetic genes and biosynthesis operon of riboflavin have been over-expressed in L. lactis, leading to kinds of folate or riboflavin that produce at greater rates. The modified biosynthetic routes of folate and riboflavin in L. lactis are used to produce both vitamins simultaneously by directed mutagenesis and selection and metabolic engineering.

1.14 Commercial forms of probiotics

It is possible to absorb probiotic organisms in two primary ways: through fermented meals and through supplements. Fermented foods may come from both dairy and vegetable sources, with yogurt and sauerkraut being the most well-known of each. Freeze-dried (lyophilized) bacteria in powder, pill, or tablet form make up probiotic supplements. For clinical effectiveness, products containing probiotic organisms must contain enough live organisms to exhibit therapeutic benefits, regardless of the way they are ingested. Both fermented foods and supplements can accomplish this feat in the same way and have pros and cons (Table 3).

Delivery system	Pros	Cons
Fermented dairy	Affordability and easy availability Ease of incorporation into daily patterns Additional nutritional benefits Enhanced bacterial survival	Contains dairy proteins and lactose Taste can be issue Not suitable when traveling Not suitable for vegans
Capsules	Ease of administration Contain no binders	Not therapeutic in upper GI tract Many contain allergenic excipients Higher cost
Tablets	Ease of administration Effective in the upper GI tract	Many contain allergenic or otherwise problematic binders and excipients (e.g., gluten) Higher cost
Powders	Effective in upper GI tract Dosage can be easily adjusted Can be incorporated into foods or drinks Contain no binders

Table 3.

Pros and cons of commercial forms of probiotics [47].

The probiotic strain that has been demonstrated to have the necessary therapeutic effect is essential to achieving successful and repeatable clinical outcomes. L. rhamnosus GG, for example, has been proven to prevent viral gastroenteritis and maintain ulcerative colitis in remission, according to research. We cannot assume that other strains of L. rhamnosus would behave in a similar fashion. In the same way, a doctor who uses the identical strain used in clinical trials should expect similar outcomes. An effect may be obtained by using a nearly similar strain.

For meals and supplements containing probiotics, the dose depends only on the quantity of live organisms present in the product, not on its composition. In clinical studies, between 107 and 1011 live bacteria per day were used. When administered in a dairy medium, it appears that 100 times less viable bacteria are required to reach the same number of live bacteria in the lower colon. In the upper GI tract, dairy appears to be a good transport medium for the bacteria, boosting their survival.

2. Nutritional requirements, health benefits and probiotics

The health advantages of probiotic dietary items have increased human appeal. Such meal is not only packed in nutrients, but also lowers the risk of many disorders [48]. “Live microbial probiotics, delivered at a sufficient quantity (106 to107 CFU/g), impart health advantages on the host,” according to FAO. The medicinal advantages of these products thereby encourage probiotic use [49]. The management of gastrointestinal and urinary tract infections was related with probiotics. Other advantages include enhancing serum cholesterol lactose tolerance levels, increasing host immunity, and preventing antibiotic diarrhea and allergy disorders related with colon cancer [50]. The growing demand for novel probiotic products nevertheless prompted the development of probiotics, these products include ice cream and baby milk powder that supply probiotics.

2.1 Health benefits

The health benefits of probiotics include increased immunological responses, relief of lactose intolerance symptoms, therapy for diarrhea, reduction in serum of cholesterol, production of vitamin, anticarcinogenic. The most important segments of world commerce have been probiotic goods in recent years which can increases annually, the growth rate ranges in 6.8% between the year 2013 and 2018, and then reached to 37.9 billion US$ in 2018. In the meantime, probiotic dairy products are one of the most advanced and a key part of the functional food business [17].

2.2 Probiotic potential of cheese

Cheese is useful because of the high pH, greater amount of fat and from its thick consistency. Cheese is a useful food for probiotics to the gastric intestinal system. The probiotic potential is studied and used as assistant cultures in various kinds of food products or therapeutic preparations a diverse number of food grade lactic acid bacteria (LAB) isolated from PDO cheese from Italian form of Castelmagno product of cheese, Italian and Argentinean cheese products [51]. Lactobacilli are isolated from milk products, in particular from cheese, and have shown a long history of safe use because these micro-organisms are widely used to develop new fermented products, milk or meat, alcoholic beverages and sourdough.

Piewngam [52], studied that the formulations of probiotics are believed to enhance human health, such as immunostimulant effects or interbacterial competition between helpful bacteria and harmful ones. The use of probiotics was seen as a potential method for the prevention and management of many infectious illnesses.

Piewngam et al [53] found a reverse linkage between human colonization with species of Bacillus and S. aureus. The researchers also detected a key mechanism through the suppression of quorum sensing through which species of Bacillus can kill S. aureus. Chung [54] described that the fengycins are the types of bacilli produced lipopeptides that are identified through the process of chromatography and mass spectrometry.

2.2.1 Efficacy of S. aureus

Another work carried out by Moraes et al. [55] has demonstrated the efficacy of S. aureus biofilms produced on titanium discs in Lactobacillus brevis and B. bifidum. The results indicated a decrease in growth of S. aureus on titanium disc when both probiotics were applied but in L. brevis strains the highest inhibitory effect was seen.

2.3 Examination of Helicobacter pylori (H. pylori) as an infection

Goderska et al. [56] examined Helicobacter pylori (H. pylori) as an infection which has been seen as difficult to treat, particularly as it has gained an increased resistance to antibiotically widely used. Probiotics in conjunction with antibiotic regimens are increasingly being used to eliminate H. pylori. In addition to the advantages of probiotic bacteria to the intestine’s probiotics have shown effective for the treatment of various bowel disorders including diarrhea; several positive effects on the stomach, including anti-Helicobacter pylori have also been documented [57].

The advantages of probiotic treatment are reduced microbial charge and host tolerance in instances with H. pylori. Several research have revealed the positive benefits of various H. pylori probiotics by reinforcing the mucosal barrier, while increasing adhesion and immunomodulation competition.

A study carried out by Lahtinen et al. [58] demonstrated that the growth of Staphylococcus aureus, often seen in systemic and peri-implant infections, has been prevented from 3 out of 38 strains of Bifidobacterium. The antibacterial activity of various probiotic Lactobacilli strains was studied by Lazarenko et al. [59]. In a model of intravaginal infection in mouse, B. bifidum (B. bifidum) was found to be largely effective against S. aureus with a substantial reduction in the amount of S. aureus cells caused by vaginal spraying. In comparison with other probiotic strains of various genera, B. Bifidum exhibited superior anti-staphylococcal efficacy.

2.4 Test model of C. albicans-infecting mouse to determine the effects of L. casei in vaginal candidiasis

In the test model of C. albicans-infecting mouse, Liao et al. [60] analyzed the effects of L. casei administration in vaginal candidiasis. The animals were inoculated with L. casei vaginally throughout 7 days for prophylactic testing. Three mice were killed, and the amount in CFU/ml was measured. The animals had C. albicans infected the vaginal cavity 2 days after the infection. The animals were treated with C. albicans in therapeutic tests and after 2 days, L. casesi was infected for 5 days. The CFU/ml number was then measured in vaginal samples. The findings suggest that prophylactic L. casei treatment might enhance vaginal mucosal immunity, increasing IL-17 production during infection. IL-23 levels had also weaker anti-inflammatory effects than those in the control group. In the therapy group, after 5 days of treatment, L. casei decreased the fungal vaginal load.

2.5 Probiotics and its advantageous effects on skin

Mottin and Suyenaga [61] described that poor skin problems might impact the quality of life of the patient due to discomfort. Human skin is made up of several fungus and symbiotic bacteria Chronic skin diseases that require lengthy treatment durations and maintenance are acne and atopic dermatitis (AD). In these situations, studies have found satisfactory outcomes without side effects using probiotics. In vitro trials indicate the potential to directly suppress acnes development by producing antibacterial proteins (bacteria) and immunomodulatory effects of probiotics, such as Streptococcus salivarius and Enterococcus faecalis. It has been demonstrated that probiotics have direct (inhibited P. acnes) and indirect (reduce the inflammatory response) advantages [62, 63].

2.6 Influence of probiotics on mental health and disease

Dinan and Cryan [64] think that the intricate bidirectional connection that happens between the brain and gut microbiota (GM) might be a novel approach to determine mental disease treatments. Several studies have found that the GM plays a substantial influence in an individual’s mood and behavior, and that it might be very useful in mental health therapy. Stress-induced physiological consequences in the stomach, such as nausea and spells of diarrhea, have a significant impact on the GM balance [65].

Psychobiotics are a novel type of probiotic that is intended to help people with psychiatric illnesses by enhancing their cognitive abilities [66] Many different gut microbial species generate a variety of mood-regulating neuromolecules, which has an impact on host physiology. GABA is produced by Bifidobacterium and Lactobacillus species, whereas serotonin is produced by Enterococcus, Escherichia, Streptococcus, and Candida species, and dopamine is produced by Bacillus species [67].

2.7 LAB with in vitro, in situ cholesterol-lowering characteristics

Cholesterol reduction is one of the most favorable properties for probiotic bacteria with lactic acid. In this work, a capability evaluation was carried out of 58 possibly probiotic bacteria containing cholesterol and bile acids for in vitro digestion and cholesterol reductions. The best-performing strains reduced cholesterol levels in broth by 42–55% and were tested in the production of cheese.

In all cheeses, the cholesterol content declined during maturation. The most significant decreases (up to 23%) were obtained by adding LB. paracasei, paracasei VC2161 and Epilithonimonas lactis BT 161 during cheese-making, all strains were present in the cheese at levels greater than 107 cfu/g up to 60 days after ripening. There was no detrimental influence on the sensory properties of cheese in the adjacent cultures. These strains with demonstrated in vitro characteristics are, therefore, ideal candidates for new probiotic formulations, and can also be utilized to make foods like dairy fermented products effective.

2.8 Probiotic strains

Probiotic microbe selection is based on safety, function, and technology, as described in the following reports. Some probiotic microorganisms are already on the market and have been thoroughly investigated. They must first be able to be produced under industrial circumstances before probiotic strains may be provided to customers. Then, throughout the storage of the crops frozen or freeze-dried as well as food items into which they are formulated, they must survive and keep their functioning. Furthermore, they need to be incorporated into plates without producing flavors or texture. For functional dietary requirements, the following aspects in relation with the probiotic should be considered: Preparing for large-scale manufacturing should be feasible, remain stable and viable for storage and use.

2.8.1 Lactobacillus rhamnosus GG

Studies have demonstrated the promotion of immunoregulatory activities by raising regulatory cytokines of interleukin (IL)-10 [68] and the induction of beta (TGF-β) transforming T-cells [69]. In fact, atopic children have proven that the gut microbiota differs from atopic ones. LGG showed a beneficial impact on atopic illness prevention while randomized clinical studies (RCTs) reveal no outcomes [70, 71].

LGG in babies with rotavirus-related diarrhea led to higher increased production of non-specific antibodies and anti-rotavirus antibodies. The neonatal evidence of necrotizing enterocolitis, improving food tolerance and prevention of pathogens colonizing intestine because of competition exclusion, preventing adhesion, and improving mucosal immunoglobulin A (IgA), has shown LGG to be effective in reducing incidences of necrotizing enterocolitis. A new retrospective 6-year cohort study on LGG in extremely small birth weight babies showed the microbiological safety of the strain [72].

2.8.2 Bb12 Bifidobacterium

Bb12 has been available on the market for over 25 years and is one of the best probiotic strains accessible for the most extensive research. It was administered to babies alone or with several different probiotic strains and has demonstrated its well-tolerated and beneficial effects [73]. The Bb12, combined with Streptococcus thermophilus Th4, has already proven that infants are well accepted in a formulation and have decreased the colic levels, irritability, and antibiotic needs for 6 months [74]. Bb12 is highly colonized because to its excellent adherence to human mucus [75]. The gut microbiome of preterm children has previously demonstrated a beneficial influence (Table 4) [70].

2.8.2.1 Bifidobacteria and colorectal cancer

Several research have looked at Bifidobacterium’s ability to prevent and/or treat colorectal cancer. The bulk of research use mouse models to reach their conclusions, and the results imply that a combination of prebiotics and bifidobacterial may minimize the incidence of carcinogen-induced malignant cells in mice [77]. For example, it has been demonstrated that Bifidobacterium animalis has anti-mutagenic activity while growing in MRS broth, effectively counteracting the action of the carcinogen 2-amino-3-methylimidazo [4, 5-f] quinolone [78]. It has also been established in vivo and in vitro that a B. longum and a B. breve strain protect DNA from carcinogen-induced damage and suppress the genotoxic impact of two separate carcinogens when evaluated in a rat model [79].

2.8.2.2 Bifidobacterium and necrotizing enterocolitis

Following regular treatment of B. breve M-16 V, recent research found a decreased incidence of necrotizing enterocolitis in premature infants [80]. Administration of B. breve M-16 V in conjunction with breast-feeding was demonstrated to be related with a decreased incidence of necrotizing enterocolitis in neonates born before 34 weeks gestation, and, while not statistically significant, a lower incidence of this disorder was found for neonates born at a gestation age of less than 28 weeks [80].

2.8.2.3 Bifidobacterium and inflammatory bowel disease

Although the precise mechanism of action is unknown, probiotic strains were shown to reduce the symptoms of inflammatory bowel disease [81]. A probiotic mixture including three Bifidobacterium strains, four Lactobacillus strains, and one S. thermophilus strain was given to patients suffering from ulcerative colitis. Fifteen of the 20 patients stayed in remission throughout the experiment, indicating that treatment of this bacterial cocktail is useful in sustaining ulcerative colitis remission (Table 5) [81, 83].

Name deposit code	Proprietary company	Product	Example
Lactobacillus acidophilus NCFM ATCC SD5221.	DuPont Danisco	Heinz	nature Toddler
Bifidobacterium animalis subsp. Lactis BB12 DSM 15954.	Chr Hansen.	Heinz	Nestle Good Start
Lactobacillus rhamnosus GG ATCC 53103. Nutramigen	Valio	Mead	Johnson Nutrition
Lactobacillus reuteri DSM 17938.	BioGaia	Nestle	NAN L.I. GOLD

Table 4.

Probiotic strains of infant formula [76].

Lactobacillus species	Bifidobacterium species
Lactobacillus acidophilus	Bifidobacterium adolescentis
L. Casei	Bifidobacterium animalis
L. crispatus	Bifidobacterium bifidum
L. gallinarum	B. breve
Lactobacillus gasseri	B. infantis
Lactobacillus johnsonii	Bifidobacterium lactis
Lactobacillus paracasei	Bifidobacterium longum
L. plantarum
Lactobacillus reuteri
Lactobacillus rhamnosus

Table 5.

Probiotic microorganisms [82].

2.8.2.4 Lactobacillus reuteri strain

L. reuteri is the probiotic strain of the probiotics L. reuteri, is a well-known probiotic. L. reuteri ATCC 55730 colonizes the stomach effectively and can reduce the occurrence of watery diarrhea associated with rotavirus. In addition, it recently showed effectiveness in the treatment of acute diarrhea with oral rehydration in children from 6 to 36 months of age [84]. A Indrio [85], research shows that L. reuteri DSM 17938 treatment has lowered stomach distension, expedited gastric emptying, and reduced regurgitation in children with normal gastroesophageal reflux. A recent study shows that this strain also has a favorable effect on the medication of baby colic if employed in several clinical studies or as a therapy for prophylaxis.

2.8.3 Saccharomyces spp. with probiotic properties

Saccharomyces genus contains several yeasts like: Saccharomyces cerevisiae that is used for the preparation of wine, bread, beer, Saccharomyces bayanus is used to produce wines, and Saccharomyces boulardii utilized in medicine as a probiotic [86].

S. boulardii is frequently advertised as a lyophilized probiotics to treat diarrhea and retains an excellent safety reputation [87]. Most reports show that S. Boulardii’s clinical advantages are reducing diarrhea duration regardless of causation and thus reducing the social and economic benefits associated with hospitalization. S. boulardii dispensation has had a positive effect on the prevention and treatment of retroactive inflammatory bowel disease and moderate symptoms of ulcerative colitis [88] patients with irritable bowel syndrome. Current Clostridium difficile pseudomembranous colitis infection can also be drastically reduced through the administration of S. boulardii regular dosages together with standard antibiotics. No malformations were reported in the previously referred examination of probiotic safety during pregnancy [89]. It should be borne in mind, however, that S. boulardii can lead to fungal diseases or localized infections in immunocompromised people or in other patients.

2.8.3.1 Escherichia coli strains with probiotic properties

Although it is known mainly for its highly virulent serotype (e.g., E. coli O157:H7), E. coli is a very common lower gut inhabitant and even a probiotic strain is known to be E. coli Nissle 1917; E. coli genus belongs in a Gram-negative family called Enterobacteriaceae (EcN). As previously mentioned, it has been proven that constipation [90] and inflammatory bowel disease were treated with other probiotics in 1917 [91]. This strain could also alleviate gastrointestinal disorder, ulcerative colitis, Crohn’s disease and even colon cancer.

2.9 Probiotic research as an advantageous facilitator in aquaculture

Researchers have previously shown that probiotic activities play a wide range in the host body (e.g., decreasing illnesses and stress, enhancing immunity, modulation of gut microbiota, nutritional assistance, improving quality of water, etc.). So, the positive effects of probiotics help to boost animal feed value and growth and improve aquaculture breeding and hatching rates. Probiotics have recently become a highly common technique in the aquaculture industry, and they are mostly isolated from fish guts. A recent study shows that Lactic acid bacteria (LAB), named Bifidobacterium, and Streptococcus are among the most common bacterial suggestions. Even though the use of probiotics in aquatic species is a relatively new concept, it has gotten a lot of interest because of its ability to influence many physiological processes.

In this study the many positive features of probiotics in aquaculture industries were proven. Probiotics are regarded as new functional agents with a potential impact on any aquatic organism’s gut microbiome. Researchers have already shown that probiotic activities play a broad spectrum in the host body, such as reducing illnesses and stress, increasing immunity, modulating gut microbiota, nutrition aid, improving water quality, etc. In addition, the positive benefits of probiotics boost feed value and growth for the animal and improve the rate of aquaculture spawning and hatching.

2.10 Probiotics and their possible uses clinical importance

Probiotics are an interesting study field that the current age needs to examine for clinical wellness. Elite properties such as anti-pathogenic activity, anti-diabetics, anti- obesity, anti-inflammatory activities, anti-cancerous activities, anti-allergies and angiogenic effects and their influence on the intelligent and central nervous system (CNS) (Figure 4).

Figure 4.

Applications of probiotic [92].

2.10.1 Probiotic anti-pathogenic action

Action of probiotics as anti-pathogenic is deemed as one of the most valuable effects in the probiotics, since the composition of the complex gut microbiota population is hindered, unlike conventional antibiotics, by disorder or change. Tejer in [93] examined the effect that probiotic substances can inhibit short-length fatty acid (SCFAs) pathogens (as acetic acids, propionic acids and lactic) on the survival activity of Salmonella enterica, Serovar typhimurium and C. difficile in a vitro scope model and postulated it. Kareem [94] investigated that SCFAs helps to maintain a colonic lumen pH that is imperative for the expression and for the metabolic rate of foreign compounds and carcinogenic substances in the gut [94]. Although it is known mainly for its highly virulent serotype (e.g., E. coli O157:H7), E. coli is a very common lower gut tenant and even a probiotic strain is known to be E. coli Nissle 1917; E. coli genus belongs in a Gram-negative family called Enterobacteriaceae (EcN). As previously mentioned, it has been proven that constipation [90] and inflammatory bowel disease were treated with other probiotics in 1917 [91]. This strain could also lessen gastrointestinal disorders like ulcerative colitis, Crohn’s disease and even colon cancer.

2.10.2 Anti-obesity activity of probiotics

The physiological actions of probiotics are important for the health of the microorganisms controlling the environment of host. Thermogenic and lipolytic reactions in most cases assist loss of weight by activation of the sympathetic nervous system. Lactobacillus gasseri BNR17, probiotic strains, have demonstrated characteristics to block the rise and therefore restrict leptin secretion in adipocyte tissues, as their major source of leptin and adiponectin. Hypocholesterolemia effects have also been shown to be present in other probiotic bacteria, such as L. casei, Lactobacillus acidophilus and B. longum.

2.10.3 The probiotics as angiogenic activity

The term angiogenesis has proven crucial for the treatment of wounds and is needed to repair damaged tissue by delineating cellular responses [95]. The angiogenic programmed includes a set of cellular processes carefully regulated by which new vessels are created by the pre-existing cell reclamation and the production of cytokines, matrix-degrading enzymes, and chemokines. Angiogenesis that is deregulated is a key influence of cancer, diabetic retinopathy and IBD including CD and UC in main human illnesses [96]. Non-pathogenic S. boulardii probiotic yeast, protective from intestinal damage and inflammation, has been observed. However, these positive benefits remain unknown about the molecular mechanisms by which probiotics mediate. Probiotics may be potentially used to alter inflammatory cytokine profiles, decrease pro-inflammatory cascade regulation, induce regulatory mechanisms in a strain-specific way, strengthen the function of epithelial barriers, reduce the visceral hypersensitivity, increase traffic in spinal afferents, and reduce stress.

2.10.4 Anti-allergic activity of probiotics

In the past, probiotics have increased awareness of its causes and preventive measures in protecting and managing allergy disorders. In-vitro studies of some probiotic products, such as Lactobacillus plantarum L67, have demonstrated that the manufacture of interleukin 12 and interferon g at your host can prevent allergic diseases (park 2016). L. plantarum 06CC2 substantially relieved allergy symptoms in another research and decreased total immunoglobulin E concentrations, ovalbumin E specific immunoglobulin and histamine in the ovalbumin-sensitive mouse sera. L. plantarum 06CC2 is reported to enhance the interferon-g and interleukin-4 secretions substantially in the cells of spleen in mice, which alleviate allergy symptoms [97]. Further investigation may be useful in assessing the anti-allergic activity and method of action of probiotics.

2.10.5 The probiotics as anti-cancerous

On the World Health Organization’s cancer data page, there were around 14 million new cancer diagnoses and approximately 8.2 million cancer-related deaths in 2012 alone. Asia, Africa, and the Americas account for more than 70% of cancer fatalities worldwide [98]. The attention has shifted to natural sources that impart anti-cancer benefits, such as probiotics, in recent years [99]. They are interested in working together to bring the illness down as well as produce a treatment with minimal or no adverse effects [100].

2.11 In vitro studies

The probiotic strains, the Lactobacillus fermentum NCIMB-5221, and -8829, have been shown to be extremely strong for the suppression and development of normal colonic cell epithelial growth by producing SCFAs in vitro studies (ferulic acid). Kahouli [101], in 2015 compared L. acidophilus ATCC 314 and L. rhamnosus ATCC 51303, that were characterized by tumorigenic activity. Probiotic strains of L. acidophilus LA102 and L. casei LC232 have shown the cytotoxic activities with two colorectal cell lines (Caco-2 and HRT-18) being in vitro anti-proliferative [102]. Although probiotics may play an important role in cancer neutralization, only in-vitro tests are confined to research. Therefore in vivo models and animals’ clinical trials must prove the potential of anti-cancer probiotics.

2.12 Efficacy of probiotics in COVID 19

The recent Xu et al. [103] trials indicated the ability of probiotics in the avoidance of secondary infections in those afflicted by COVID 19. Some COVID-19 individuals suffered from microbial intestinal dysbiosis. All patients need to examine their dietary and gastrointestinal functioning. The regulation of the stability of gut microbiota and the decreasing probability of secondarily infected bacterial translocation should be supplemented with nutrition and application of probiotics.

2.12.1 Probiotics for COVID prevention

Probiotic medicines against viruses which lead to respiratory tract infections are proposed in the last two decades as antimicrobial agents. There are numerous conceivable action mechanisms to increase breath-probiotic activity; the modulation of the innate immune system and better immune response are nevertheless most probable. According to earlier research of certain viral illnesses, preventing infectious diseases can be achieved by increasing and activating human immunological activity by healthy, equilibrated meals and administrative complements such as vitamins, minerals, fiber, and probiotics [104].

Live microorganisms which provide a sufficient intake of health advantages, including an increase in immune activity and removal of respiratory tract diseases, are probiotics. Probiotics can obviously lower the prevalence and severity of illnesses, showing their promise to cure or prevent COVID-19. To manage viral infection, it is important to understand the immune cell activation, cytokine profile and immunological regulation. The preservation of the human GI or lung microbiota might help prevent COVID-19, as dysbiosis plays an essential role in people’s vulnerability to infectious illnesses The potential preventative and therapeutic impact of probiotics against SARS-CoV-2 infection should be examined in in vitro and clinical investigations.

3. Isolation of probiotic microorganism

The initial process that was carried out for the isolation of probiotic bacteria is to keep the sample selectively before the step of incubation in suitable conditions. Many probiotics are anaerobic; hence, the samples then placed into anaerobic conditions immediately (within 3 h). The samples should be immediately homogenized, diluted, and cultivated in selective media Several mediums were developed to isolate bifidobacteria and lactobacilli either electively or selectively. As a source of isolation for probiotic bacteria were utilized milk fermented products (curd, buttermilk, cheese) and vegetable pickles. Direct plating and enrichment methods were used for the isolation of MRS agar and MRS broth, respectively. These samples were diluted in 9 ml of saline water serially up to 10-4 (0.89% NaCl) with a spread of semi-solid MRS on Petri flat plates. The inoculates were then incubated at room temperature for 2 days to examine and control microbial colonies that grown on medium (without inoculation).

3.1 Spreading

Appropriate sample dilutions were made, and a concentration of just 50 PI-J was dispersed on MRS-agar plates. For 48 h, each plate was kept at 37°C in a static incubator.

3.2 Streaking

A single bacterial colony was plucked with a sanitized loop and streaked over plates to get isolated colonies. The plates were then put in a static incubator at 37°C for 48 h.

3.3 Characterization of bacterial isolates

3.3.1 Morphological tests

These morphological tests were performed to identify bacterium isolates. These tests are as follows:

3.3.2 Gram’s staining

This staining was used to discriminate between gram-positive and gram-negative microorganisms. Gram staining may be used to distinguish the morphology of bacteria, such as bacillus or coccus.

A neat and clean glass slide was prepared initially for gram’s staining, and a thin smear of a single colony was created. The slide was then air dried. After that, the slip was fixed by running it through the flame 5–6 times. The prepared smear was then coated with crystal violet for 30–60 s. To remove any remaining discoloration from the slide, distilled water was utilized. For 30–60 s, Gram’s iodine solution was applied to the smear. Following that, the initial stain was removed with alcohol for 30 s. The slide was drained with tap or distilled water before applying the secondary stain safranin for 1 min. Rinse the slide once again with distilled water. To dry the slide, blotting paper was employed. The slide was then examined under low and high magnification towards the end. Microscope power, i.e., at IOOX in oil immersion [105].

3.3.3 Endospore staining

• Endospore staining is used to distinguish spore-forming bacteria from non-spore-forming bacteria.

• To stain endospores, a crisp and clean slide was obtained, and a thin smear was created using an isolated culture of bacteria. The smear was then air dried. The smudge was fixed by passing it through the flame 5–6 times. Blotting paper was put across each slide after it had been fixated.

The malachite green stain was then put on blotting paper over steam for 15–20 min. After the slide had cooled to room temperature, the blotting paper was removed, and the slide was washed with distilled water for 30 s. The slide was then treated with safranin for about 2 min before being rinsed with deionized water. To dry the slide, blotting paper was used. Finally, the slide was examined under a microscope with low and high magnification powers, i.e., at 100× with oil immersion [105].

3.3.4 Motility test

A motility test is a test that is used to determine if bacteria are motile or not.

Semi-solid medium was necessary for this purpose. Tryptone log, 5 g yeast extract, 13 g agar, and NaCl 5(g) were used to make the medium, which was then diluted in 1000 ml distilled water.

After that, 10 ml of it was poured into a variety of test tubes, cotton plugs were used to seal the test tubes’ mouths, and it was sterilized in an autoclave at 121°C for 15 min. Finally, the medium was allowed to harden vertically. The medium was infected using a red-hot inoculating needle and then incubated at 37°C for 24 h [105].

3.3.5 Biochemical characterization

3.3.6 Catalase test

This test was carried out to determine the capacity of microorganisms to digest hydrogen peroxide (1-1202). A nice and clean glass slide was used to perform the catalase test. In the center of the slide, one drop of water was placed. Using an inoculating loop, I took some isolate culture and mixed it with water. I applied 2 drops of hydrogen peroxide to it and witnessed the results [105].

3.3.7 Casein hydrolysis test

Skim milk agar medium was made for the casein test by combining 2 g tryptone, 1 g yeast extract, 6 g agar, 4 g glucose, and 4 g skim milk in 400 ml of distilled water. The media was steam sterilized in an autoclave for about 15 min at 121°C, and the petri plates were poured under sterilized conditions. The isolated colony of each bacterium was then streaked in the middle of the petri dish under sterilized conditions. Each plate was placed in the incubator at 37°C for 24 h before the change was detected in each petri dish [105].

3.3.8 Carbohydrate fermentation test

The goal of this test is to confirm the microorganisms’ ability to ferment carbohydrates via gas and acid [105].

3.3.9 Glucose fermentation

The medium for this experiment was phenol red broth. 5 g NaCl, 0.018 g phenol red, 10 g peptone, and 5 g glucose were dissolved in 1 liter of distilled water to make phenol red broth. The medium was then autoclaved for 15 min. The bacterial culture was then injected in the medium. For 24 h, test tubes were put in an incubator set to 37°C. If the color goes from red to yellow, the result is good. To observe gas generation, a Durham tube was inserted in each test tube [105].

3.3.10 Lactose fermentation

The medium for the lactose fermentation test was made by combining 0.018 g phenol red, 5 g sodium chloride, and 10 g peptone 5-gram lactose in 1000 milliliter deionized water. The medium was then autoclaved sterilized. The medium was then injected with a bacterial culture. After that, each test tube was placed in the static incubator overnight at 37°C. The presence of yellow suggests a favorable outcome. Durham’s tube was used to monitor gas output.

3.3.11 Sucrose fermentation

The sucrose fermentation test was performed to determine whether or not a microorganism had the capacity to ferment sugar. To conduct this test, the following substances were dissolved in 1000 ml of distilled water: 10 g peptone, 0.018 g phenol red, 5 g sodium chloride, and 5 g sucrose. The medium was then sterilized in an autoclave at 121°C for 15 min. After that, the medium was injected with bacterial culture. For 24 h, the test tubes were put in an incubator set to 37°C. If a yellow tint develops, it indicates that fermentation is taking place. Durham tubes were inserted in each test tube to observe gas generation [105].

3.3.12 Probiotic properties of isolates

The following are the primary selection criteria for determining the probiotic characteristics of bacterial isolates.

3.3.12.1 NaCl tolerance test

Bacterial isolates were exposed to various NaCl concentrations to determine their tolerance range. For this, MRS broth containing 1–4% NaCl was injected with 0.1 ml of each bacterial suspension’s stimulated bacterial culture and incubated at 37°C for 48 h. The growth rate of the organism was estimated by obtaining the O.D. in a spectrophotometer at 600 nm.

3.3.12.2 Antibiotic sensitivity test

This experiment was carried out to determine antibiotic sensitivity. Antibiotics such as amoxicillin, penicillin, erythromycin, ciprofloxacin, gentamicin, and cefixime were used for this. First, the freshly cultured bacterial isolates were dispersed over the sterile filled Nutrient Agar plates. The antibiotic discs were then put on petri plates at identical distances and incubated at 37°C for 18 h. The creation of a clean zone surrounding the discs was seen after 18 h.

3.3.13 Molecular characterization of bacterial isolates

The following steps were used to characterize isolated bacterial isolates molecularly.

3.3.13.1 Isolation of genomic DNA

In 20 ml of autoclaved LB broth, isolated colonies of chosen bacteria were inoculated. For 18 h, these falcons were kept at 37°C in an incubator that was constantly shaken. After incubating for 18 h, the broth culture was put into an Eppendorf tube. That Eppendorf was centrifuged for 5 min at 40°C and 6500 rpm. The particle was kept, but the supernatant was discarded. Pellet was thoroughly washed with 200 PI of TEN buffer and mixed with a vortex.

This combination has been centrifuged for 5 min at 40°C and 6500 rpm. The supernatant was discarded once more. The pellets were preserved for future use. Following that, 100,111 of SET buffer was put into an Eppendorf and the pellet was well mixed using a vortex. The Eppendorf was then filled with 100 gl lysozyme and placed in the incubator at 37°C for 30 min.

Following that, 5 PI of 25% SDS solution and 100 PI of TEN buffers were added. The Eppendorf tube was gently inverted many times until lysis occurred, and then incubated at 600°C for 15 min. After withdrawing the Eppendorf from the incubator, it was allowed to cool at ambient temperature before being filled with 5 gl of 5 M NaCl. The mixture was treated with an equal proportion of chloroform and buffered phenol (l:l). Eppendorf was centrifuged at 40°C for 1 min at a speed of 6500 rpm. Supernatant was removed once more and transferred to a fresh Eppendorf tube. The DNA was then precipitated by adding twice the volume of absolute ethanol (100%) that had to be ice cold. Overnight, the Eppendorf was chilled.

The next day, Eppendorf was centrifuged for 5 min at 6500 rpm (40°C). The supernatant had been decanted. The pellet was rinsed with 70% ethanol. The Eppendorf was properly air dried. TE buffer (50 microliter) was added, and the DNA was kept at 20°C for future use.

3.3.13.2 Gel electrophoresis

Gel electrophoresis was used to determine if the treated materials contained isolated genomic DNA or not. 1% agarose gel was made for this purpose by dissolving 1 g of powdered agarose in 2% 50× TAE buffer. 2 ml of 50× TAE buffer was dissolved in 98 ml of autoclaved distilled water to get the 2% solution. Unless the agarose and buffer solution was correctly mixed, it was heated in the microwave for over 2 min. After cooling to room temperature, ethidium bromide 2 PI was added. When the temperature was reduced to 600°C, the gel was gently poured into the gel casting tray. Before pouring the gel, the comb was placed in the gel casting tray. The gel plate was put on a level surface to make the gel smooth and consistent. A 20 PI sample was obtained in an Eppendorf tube and 5 gl of 1× loading dye was applied to it. The sample was stored on ice during processing. After solidification, the comb was carefully removed from the tray, and the test sample was placed in each well. For over 40 min, electrophoresis was performed at 80 volts. A DNA ladder (10 Kb) was put into one well.

3.3.13.3 Polymerase chain reaction

Ferment was used as a PCR reagent in the PCR of 16S rDNA. The samples were put into a Thermo cycler (Progene, Techne) that was set for initial denaturation at 940°C for 20 min, melting at 940°C and annealing at 5200, primer extension at 720°C for 60 s each, for a total of 35 PCR cycles (Figure 5).

Figure 5.

PCR steps.

The last extension at 720°C lasted 10 min, and the ultimate storage temperature was set to 40°C for the maximum term.

3.3.13.4 Gel electrophoresis of amplified product

1% agarose gel was created for this purpose by combining 1 g of powdered agarose with 2% of 50× TAE buffer. Amplified items were placed in the appropriate wells. It was ran at 80 volts for 40 min. The gel was then examined for the presence of amplified products using a UV trans-illuminator.

3.3.13.5 Purification of PCR product from agarose gel

After amplification, the required DNA band was sliced, and the net weight of the agarose gel containing the DNA band was calculated. After that, an equivalent volume of binding buffer was added and the gel was incubated at 50°C until fully melted. The sample was then transferred to a column. The column was centrifuged at 10,000 rpm for 1 min, then washed with 750 gl of wash buffer and centrifuged again for 1 min. The flow through was removed, and the column was washed one more with 750 gl of was buffer. For 1 min, the column was centrifuged at 10,000 rpm. Flow through was discarded once again, the column was moved to a fresh micro centrifuge tube, and 30–50 PI elution buffer was added. It is then permissible to centrifuge for 2 min at 10,000 rpm for 30 s and store at −200°C for future use.

3.3.13.6 Sequencing

Following purification of the PCR products with the-Invitrogen gene clean, the samples were forwarded to the laboratory for 16S rRNA sequencing.

3.4 Identification

The initial stage in the identification of potential probiotics is to identify microorganisms inside the GIT or in dietary sources. Only a tiny proportion of microorganisms can be cultivated in various habitats in culture [106]. The taxonomic categorization characterized as the process of cataloging that was based on a polyphasic approach [107]. Phenotypical techniques used to identify microorganisms have historically been employed. For many decades, the taxonomy depends on significantly on the kind of sugar fermentation and fermentation products. The probiotics were therefore categorized largely as LAB. The method of choice today is 16S rRNA gene analysis. Microbiologists have employed this conserved region for phylogenetic categorization for the past two decades, and the relatedness of species is inferred by comparing their sequences in publicly available databases. To detect bacterial communities from gutorecological sources, 16S rRNA gene analysis was coupled with other techniques.

The amplified 16S rDNA may relate to PAGE by means of the hybridization using fluorescent oligonucleotide probes (fluorescence in situ hybridization) or chemical denaturation using restricting enzymes (T-RFLP) with a specific 16S. However, in comparison with the bacterial Genome having base pair of 30,000–40,000, the 16S rDNA segment is exceedingly tiny (1500 bp).

3.5 Characterization

The two most significant probiotic-taxa are Lactobacillus and Bifidobacterium species in processed dairy products. When eaten, sufficient metabolically active bacteria are required to penetrate the GIT barrier and have transitory impact in GIT. This is essential since some writers have demonstrated the positive benefits of dead probiotics [108]. GIT has the challenge to survive on GIT with the potential to withstand with extremely low pH of about 1.5, availability of gastric enzymes, bile salts and other bowel enzymes [109]. Different in vitro tests to imitate these stress conditions have been devised.

3.6 Identification and characterization of probiotics

Isolate were identified by gram staining, endospore stain, catalase testing, and carbohydrate fermentation test. The growth and survivability of the stomach and small intestines is part of this. The stability of these properties following ingestion must thus be tested to verify that they are maintained in the host. Therefore, tests of acid and bile tolerance should include early screening and selection of probiotic strains.

Classical physiological and biochemical assays are not efficient for analyzing and quickly identifying microbial communities, as the bacterial population typically has comparable nutrient requirements and develops under similar environmental circumstances. Thus, it may often be difficult to clearly identify the species using simple phenotypic criteria. New possibilities for defining strains of fermented milk items have been developed using molecular methods. The 16S rDNA assays are fast, and cheap to detect the microbial species of yeast, acetic acid, and of some Gram-positive bacteria. Among PCR-based techniques is easy and cost effective. The chosen strain for diverse probiotic characteristics was characterized. These comprise the susceptibility analysis of antibiotics, the capacity to create bioactive metabolites and acid sensitivity tests for their antibiotically resistance potential.

3.6.1 Antibiotic susceptibility test

Several antibiotics were employed at different concentrations, ranging from 25 μg, 50 μg, 100 μg, 200 μg, 250 μg and 500 μg/ml agar medium, including the penicillin G, tetracyclines, gentamycin, vancomycin, and streptomycin. Lactobacillus has been streaked across an agar plate over the overnight culture. It was aerobically incubated at period of 48 h for 37°C, controlled and checked for lack of growth.

3.6.2 Antimicrobial production

Bilkova et al. have evaluated the antimicrobial production of the chosen isolate. As an indicator strain E. coli was utilized. The supernatant culture was caught at the time period of different intervals consist of 21, 24, 27, 30 h of inoculation following intervals. Above supernatant the proteinase, followed by heat treatment, was centrifuged, and digested. It was put into 200 μl wells in E. coli lawn containing plates and incubated with control (sterilized water) for 37°C for 48 h to check for the presence of the indicator strain growth inhibition zone.

3.6.3 Acid sensitivity test

In MRS broth it was controlled by examining the capacity of isolated bacteria to grow at the acidic environment in different pH. After 24 h incubation at 37°C at 150 rpm, OD values at 600 nm were measured. The bacterial growth was compared with growth in the pH 7 MRS broth by determining its acid sensitivity.

3.7 Studies demonstrating characterization and identification of probiotic isolates

3.7.1 Isolation from cheese sample

Ward and Timmins [110] published another investigation which found that three strains isolated from cheese samples having Lactobacillus casei, Lactobacillus paracasei and Lactobacillus rhamnosus were distinguished by PCR. In MRS agar samples, 63 isolates were cultivated and evaluated with sugar fermentation phenotypes. These isolates then belonged to the casei group. PCR primers were developed for 16S rRNA gene preserved areas and particular fragments were amplified with PCR. The agarose gel separated the amplified products from the data base and retrieved their gene sequences. These three distinct strains in the region VI of 16S rRNA have given unique amplified products. At the end of the process, a product containing the 16S rRNA Lt. paracasei gene was discovered in 51 of 63 isolates and a product containing the 16S rRNA gene L rhamnosus was given in 12 of 63 by primers, whereas a product containing the L. casei primers was not detected.

3.7.2 Bacterial viability protection during in vitro gastric transit simulation in fresh cheese from Argentina

Vinderola et al. [111] examined fresh cheese from Argentina a type of soft rindless cheese that have a 12-day maturation at 5°C before it is sold on the market. The following parameters are shown in this cheese: 5.29 pH, 58% humidity (w/w), 12% fat (w/w), 23% proteins (w/w), 0.9% salt (w/k), ashes (w/w), 40.8% dry matter (w/w) and 0.6% calcium (w/w). This product has shown suitable for the preservation and consumption of probiotic microorganisms. It gives a certain level of bacterial viability protection during in vitro gastric transit simulation.

3.7.3 Counts of L. acidophilus to improve the flavor, and texture

Kasimoglu et al. [112] demonstrated that the strain of L. acidophilus may be utilized in the production of the probiotic Turkish white cheese. To make health claims, the finalize counts of L. acidophilus were more than the minimum (107 Cfu g⁻¹) needed. L. acidophilus may also be employed in the production of high levels of proteolysis to improve the flavor, and texture. In addition, vacuum-packed probiotic cheese was demonstrated to be more acceptable after salting than the same cheese kept in salt. The recommended way of preserving probiotic Turkish white cheeses is thus the vacuum packing.

3.7.4 Probiotics in cheddar cheese

Phillips [113] researched about probiotic cheddar cheese. Six samples of this Cheese were produced with various mixtures of probiotic flora that was commercially available. Every supply, the specie of Bifidobacterium, Lactobacillus acidophilus and either Lactobacillus casei, paracasei, or rhamnosus have been present in cheeses. They described adequate viable counts and a beneficial impact on the consistency and sensory characteristics of cheeses. Cottage cheese has a suitable profile for the inclusion of probiotic cells. Moreover, cottage cheeses are, due to their low-fat content, a healthier alternative to many other cheeses.

3.8 Isolation of bacterial strains done by marketed foods and drugs

Liu et al. [114] assesses 41 lactic acid strains, 36 of which have been identified and obtained from the commercial produced milk and pharmaceutical products, and 5 forms of probiotics were assesses that were obtained from China. These samples then incubate in the incubator for the period of 2 days in the conditions of anaerobically provided medium at the temperature of 37°, so different colonies have been morphologically chosen and that were classified as rods or cocci under a light microscope. After being plated on suitable agar plates, pure colonies have been isolated. The conventional microbiological techniques of colony appearance, gram staining, oxidase, and catalase reactions were initially used for all isolated isolates. Amplification of the PCR and further sequencing of 16S rDNA at genus level have been carried out. For identification purposes, universal primers 27F and 1492RP were employed. AGAROSE Gel was then separated and purified by electrophoresis by 1.5% (w/v). ABI DNA Sequencer 3730 was used to the purified products. The alignment of 16S rDNA was done by BLAST.

3.9 Isolation of probiotics from milk and fermented derivatives

Mishra and Sharma [115] isolated probiotics from milk and its fermented derivatives such as buttermilk, curd, and cheese. Following direct plating on MRS agar, the colonies were serially diluted in saline water (0.89% NaCl) up to 10-4 before being distributed over MRS agar plates and cultured for 48 h. Purified isolate colonies were streaked on agar plates and morphological features of colonies were used to identify them. Gram staining, catalase test, and carbohydrate fermentation test were used for physiological characterization, and the results revealed five types of isolates, with bacteria isolated from curd and buttermilk samples being gram positive, catalase negative, and capable of fermenting glucose and mannitol without manufacturing gas. The isolates from the cheese and milk samples were determined to be gram negative, thus they were not included in the study. The isolates from the curd sample were further examined for antibacterial activity and antibiotic susceptibility, with positive findings indicating that they belonged to the Lactobacillus casei genus.

Lactobacilli isolated from traditional milk products of 17 sample samples known in Azerbaijan as tvorog curd cheese. 17 samples have been taken of tvorog, followed by 1 g suspension of each sample in solution of saline. The MRS broth was then added 500 μl of suspension and then it was incubated for the period of 48 h at 37°C. In the concentration of 10.0 ml PBS buffer (pH = 3), 1.0 ml of each cultivation of the enriched culture was incubated for 3 h to detect lactobacilli resistance to severe stomach conditions. The 10 ml of broth was added and incubating at 37°C for the period of 4 h resuscitated pH-resistant bacteria after centrifugation. Another test was performed to determine the bile salt resistance for bacteria. The bacteria that were acid resistant were injected in MRS broth and incubated for 4 h at 37°C. The dilutions were placed on MRS agar plates and incubated for the period of 24–48 h at 37°C. In 10 ml MRS broth, different colonies were selected and cultivated. The isolates were first assessed using gram staining and the morphology of cell. After that, the isolates were stored at −70°C in MRS broth with 10% skim milk and 25% glycerol. All these species have antibacterial properties against the indicator bacterium. Isolation of antibacterial samples by tvorog curd cheese, to determine acid and bile resistant lactobacilli strains, that was identified by 16 s rDNA as L. plantarum, L. casei, and L. rhomnos.

Tavakoli [116] gathered five specimens of Koozeh from remote area of Mazandaran province. The sample (30 g) has been homogenized and then incubated for the period of 24 h at 37°C and inoculated in 300 ml of MRS Broth. In the following stage, the pellet was resuspended to be 10 ml phosphate-buffered (PBS) in a 2.5 pH-adjusted phosphate-buffered Saline (PPS), then placed on an MRS agar medium. The plate was incubated in anaerobic circumstances at 37°C for 24–48 h. Colonies of all morphologies were gathered and purified by culture on the same medium. Gram positive, catalase negative isolates were regarded as a presumptive LAB following gramme staining and catalase reactions, which was kept at −80°C in 15% glycerol. The different isolates were examined based on colony morphology (form, superficie and color), cell morphology (form and size), and the biochemical properties of phenotypes and lactobacillus. They were tested. Jayne Williams in 1977 described or explained that the genus Lactobacillus that depends on the morphological state, and biochemical features, has been deemed to include eight bacterial isolates. For the sequencing, the typical 16S rDNA amplicons were picked for each of the several profiles. The homology of the sequence sequences was more than 95% for four distinct Lactic acid bacteria species [117] in terms of molecular identity comparisons. There, L. plantarum, L. casei, L. pentosus, and L. fermentum were as follows.

3.10 Probiotic potential of LAB isolates

This study examined the propensity to promote the activity of β-galactosidase, CSH, generation of hydrogen peroxide, antibiotic sensitivity, and pathogenic microbes, in vitro, of eight strains of lactobacillus isolated from Iranian conventional cheese, “koozeh,” The results demonstrate that all lactic strains are powerful probiotics in developing novel formulations for the design of health-promoting functional food items. Analysis showed that the best of test probiotics among the tested are L. fermentum named MT. ZH893 and MT. ZH993 and MT. ZH593 L. plantarum. Lactobacillus acidophilus is an initially isolated bacterial bacterium economically important and was named Bacillus acidophilus from human gastrointestinal tract in 1900. During the development of technologies for the identification of bacteria L acidophilus is the typical species of a very varied and heterogeneous Lactobacillus group that has undergone several taxonomical revisions. The characterization of L. acidophilus has suffered with misrepresentation because of the difficulties of distinguishing phenotypically identical species by morphologic and different biochemical techniques. In comparison, L. acidophilus sensu stricto is currently one of the most characterized species of Lactobacillus and it is used as a supplement for probiotic that is present in functional foods. The source of L. Acidophilus strains is established, L. acidophilus historically and now misidentified, and the probiotic has genomic, and physiological features.

Another study conducted by Maged in [118] identified 93 lactic acid bacteria (LAB) from the local and fermented milk samples of 13 in number that were gathered, including fresh raw milk, crude frozen milk, and different types of cheese like (fresh, salty, cooked) yogurt stirred, (shrimp milk) yogurt, and butter. The LAB counts were greater under circumstances of microaerobic incubation than in conditions of aerobics. On the MRS solid medium surface, the colony morphologies of the isolates were seen; the colors of colonies observed were white to pale creamy with circular shape and the width that ranged from a diameter of 0.5–4 mm. About 92.47% strains were gram-positive. Catalase and cytochrome oxidase activities varied in isolated isolates, as seen that 71% were unfavorable for the activity of catalase and cytochrome oxidase production were negligible about 72%. Also, 96% of isolates were nonmotile and the motility of isolated strains varied. All the isolated strains were amplified with the 16S rDNA gene. Then by the usage of amplicon it was being sequenced and purified. The 46 distinct genera’s nucleotide sequence, i.e., Enterococcus, Lactococcus, Lactobacillus, Streptococcus and Weissella, are in line with 16S rDNA sequences from 14 species. The nine strains of the genus L. acidophilus, L. casei, L. paracasei, L. plantaarum and L. futsaii were analyzed to indicate that there could be two strains named (i.e., Hadhramaut4 and Musallam2), four strains named (i.e. MSJ 1, BgShn3, MasaLam7, and Dwan5), one strain namely (i.e. NMBM1), and another strain, (i.e., EMBM2), respectively.

Ten condensed yogurts imported from China were studied by Qian [119]. In the MRS medium, the strains of Lactobacillus grew at a temperature of 37°C at 16–24 h. Brain Heart Infusion Gardnerella vaginalis (ATCC49145) with yeast extract supplement (1%), maltose (0.1%), glucose (0.1%), and horse serum (10%) (BHIS) at 37°C for 24 h under anaerobic circumstances. It was cultured on Luria- Bertani Medium (LB) for 12 h at a temperature of 37°C (ATCC 25922). Streaking is done by MRS agar and broth and enriching them for the strains of Lactobacillus. Gram-positive colonization’s were chosen and inoculated in a broth of MRS with characteristic Lactobacillus shape, white in color and fruity fragrance. Then genetic investigation with the use of PCR and 16S rDNA sequencing has validated and identified isolates. A bacterial DNA isolation kit was used to extract the genomic DNA from the strains of Lactobacillus. The 16S rDNA genes were amplified using the universal PCR primers 27F (AGAGTTGATCGGCTCAG) and 1492R (TACGGC TACCTTGTAGACTT).

3.11 Study of phylogenetic analysis of probiotics

Hajigholizadeh et al. [120] isolating LAB from traditional cheeses and characterizing them. In 225 ml the quantity of peptone water is about 0.1% w/v was added and then mix the 25 g of each sample of cheese. Then dilute the sample of cheese in a suspension containing 2% of the sodium citrate and then grown on MRS agars and incubated for 1–2 days at a temperature of 37°C under anaerobic and aerobic conditions. From each plate of cultivation, the 3–4 distinct colonies were picked randomly. Gram staining microscopically inspected, and the catalase analysis was carried out. Molecular and antibacterial characterization is analyzed and kept in a test tube that contain 15% (v/v) glycerol at −20°C. Extraction and amplification of bacterial genomic DNA. Different kinds of bacteria colonies emerged on MRS agar surface following screening and phenotypic characterization. Screenings were performed on different 60 MRS agar plates that have tiny, round, matt, and white colonies, it was performed with 70 different biochemical characteristics of LAB showcasing the gram-positive and the catalase negative, including cocci, or in shape rods.

3.11.1 PCR and RFLPs

Koohestani [121] study based upon the spot-on-the-lawn approach was performed to assess the performance regarding antibacterial activity of 8 LAB isolates with various patterns of RFLP. For all 70 bacterial isolates, a DNA fragment contains the size of 1.540 bp has been enlarged as shown in Figure 6. Three distinct digestive patterns were shown in the RFLP PCR product analysis (patterns I–III) shown in Figure 6. Out of 70 isolates, RFLP pattern I was shown in amplified fragments from 16 rRNA from 63 (90%) isolates. The RFLP patterns I and III were all RFLP patterns discovered with the HinfI named endonuclease enzyme, and each with two and five isolates.

Figure 6.

Staphylococcus planktonic form and biofilm were inhibited by cell free supernatant of Lactobacillus casei [121].

LAB isolates were identified based on the generation of the phylogenetic trees from the sample of cheese named Enterococcus subsp., Lb. lactis, Lb. farciminis, and Lb. paracasei Enterococcus subspecies was made up of majority of the LAB (90%) in that RFLPs isolates named [2, 97, 122, 123] isolate it. RFLP pattern II was classified jointly in Isolates namely 14 and 32. The RFLP pattern III was identical to two isolates 22 and 44; nevertheless, these two isolates were grouped into two different clusters based on a phylogenetic tree. The most common LAB in traditional cheeses in this research were enterococcus subsp.

3.12 Enterococcus safety evaluation and probiotic potential by genomic analysis

Enterococci are the ordinary people of the human and animal gastrointestinal system. Recently, the selection of helpful microorganisms by bacteriocins was a novel probiotic characteristic [124]. The 1st OB14 strain and 2nd OB15 strain of lactic acid were isolated and identified as E. faecalis from Testori and Rigouta typical Tunisian fermented milk products. These novel isolates have been examined for the character of the gastrointestinal tract and proven tolerant to severe circumstances. They were moderate biofilm makers that increase the trans epithelial resistance and they can attach to the cells of intestines to reinforce the barrier. Different antibiotics susceptibility is seen includes Ampicillin, vancomycin, gentamicin, and erythromycin and the evidence shows the susceptibility of E. faecalis OB14 and for OB15 to essential ampicillin and vancomycin clinical drugs. The tetracycline resistance existence and the presence of cytolysin genes in E. faecalis OB14 1st strain, nevertheless, was found in the Whole Genome Sequencing (WGS). Hierarchical cluster analysis reveals the tight connection between E. faecalis OB15 and E. faecalis Symbioflor 1 against OB14. E. faecalis OB15 looks therefore trustworthy as a probiotic food or feed business for future growth.

3.13 Study of strain of probiotic potential by isolation and identification

Samples of Ezine type of cheese have been mixed and attenuated into the solution of Ringer and plated with aerobic incubation at 37°C at a species of kanamycin aesculin azide agar for 48 h. After incubation, colonies exhibiting Enterococcus-typical shape were randomly chosen and spotted using sterile toothpicks on plates containing agar. A total of 114 colonies with characteristic Enterococcal shape were transferred to BHI Agar in KAA agar. It was observed that 84 colony areas greater than 10 mm versus indicator strains displayed inhibitory zones (results not shown). PMD74 was identified as the strongest antibacterial activity for additional tests. Among these colonies. The distinctive characteristics of the strain are compatible with general characteristics. The isolate has been identified as an Enterococcus lactis strain, according to the sequence of 16S rRNA, undertaken to ensure molecular identification.

The latest investigation showed that Ezine (PDO), which consists of nonstarter Turkish white long-ripened cheese, serves as an isolation source for new enterococcal strains. This is the first analysis on E. lactis isolation in Turkey, to the best of our understanding. E. Lactis is a probiotic candidate because of the results such as strong strain tolerance to GI tract virtual circumstances, other physiological features, and remarkable antibacterial activity to both near relatives and dietary-borne pathogens.

3.14 Commercial interest in probiotics and sensory evaluation of food matrix

Commercial interests also exist for the idea of probiotic food, as is shown in the range of probiotic products accessible in supermarkets and specialized stores, that make up a major portion of the functional food market. Singh (2011) observed that various author has demonstrated that frequent ingestion of live probiotic microbes might be useful to improve lactose tolerability, reduce cholesterol levels. It was observed that probiotics may directly or indirectly affect the intestines by modifying the physiology of Endogenous Microflora or Immune System, as colonization of some strains can decrease the severity of acute diarrhea in children. Probiotics have a positive impact on the intestinal microbiology, including antagonistic effects, competition for immunological effects and improved infections resistance.

The usage of bacteria at the expense of potentially dangerous bacterial proliferation therefore encourages the proliferation of beneficial bacteria which enhances the natural defensive systems of the host. In fermented milk products, the application of probiotic bacteria has been extensively explored due to problems in maintaining the vitality of these organisms during cooling storage. The survival of probiotic bacteria in fermented dairy product may be influenced by factors such as acidity and dissolved oxygen and species interactions, inoculation techniques and stock conditions.

The quantity of viable bacteria in the intestines and the level of pH that is low in stomach leads to the limit the survival of probiotics. Furthermore, there are still numerous difficulties with the poor viability of probiotic bacteria in milk meals. In fermented dairy products there are several variables that impact the survivability of probiotics: acidity, pH and hydrogen peroxide, dissolved concentrations, stock-temperature, interaction in products with other microorganisms, lactic and acetic acid concentration, and protein concentration [125].

4. Some highlights on the LAB and possibilities for the future

Without initially securing food safety, there can be no feeling of global security. This involves, among others, supplying the world’s thriving population with safe and healthy food. The future is undoubtedly hopeful given the tremendous potential in the utilization of LABs as probiotics. One topic now being explored, for example, is the examination using whole genome sequence technology of probiotic propensities. Among other effects, the functions of probiotic LABs will be improved, and data may be utilized to further modify LAB genes [126]. More study is being undertaken on their usage as functional food components and will expand soon. The effects of probiotic LABs on the cells of breast and likes to have previously been researched to bridge the gap between the world’s food, medicinal and health industries. However, it must be noted that the safety for the improvement of food is not a probiotic feature of lactic acid bacteria (LAB).

Over the recent decade, numerous researches have been carried out examining the molecular foundation for possible probiotic characteristics of prospective LAB strains and their products, drastically enhancing our biological understanding [127]. These findings have been and still can form the foundation for important in vitro and in vivo investigations for food, biomedical and pharmaceutical specialists. These studies are crucial. Preliminary findings from current study at the Northeastern Agricultural University’s Key Laboratory of Dairy Science (KLDS).

Based on the research provided in this review, various strain combinations may thus be hypothetically utilized to evaluate the attenuation effects on gut-microbiota with respect to obesity and T2D of probiotic LABs and Bifidobacterium species. To create new anti-obesity foods and dairy products, similar research evaluating the effect of various strains on the gut microbiota may be carried out. Such research might open fresh and new medicinal and food goods pipelines with huge industrial uses.

5. Conclusion

Probiotics mean live organisms that have positive effects on the health of host. The aim of current study was to isolate probiotic bacteria from different non-dairy products. The total of 1 l samples was collected from different areas and from these samples 10 bacterial strains were isolated. All of these were characterized based on morphological, biochemical tests and 16S rRNA ribotyping. Probiotics are helpful bacteria that are catalase negative and help to restore microbial equilibrium in both people and animals’ guts. Lactobacillus was the first probiotic to show a positive influence on health. These “Good Microorganisms” can be gotten not only from non-dairy products, but also from processed dairy products such as. They improve epithelial barriers, increase adherence to gut mucosa and microbial adhesion, produce antimicrobial compounds, and regulate immune systems, and can be used as food supplements to treat various gastrointestinal tract diseases, as well as in research studies to develop commercial probiotic foods. Probiotics have health advantages such as improved immune responses, easing of lactose intolerance symptoms, diarrhea treatment, cholesterol decrease in serum, vitamin generation, and anticarcinogenic properties. Probiotics serve a variety of roles in the host body (e.g., decreasing illnesses and stress, enhancing immunity, modulation of gut microbiota, nutritional assistance, improving quality of water, etc.). As a result, the beneficial effects of probiotics contribute to increased animal feed value and growth, as well as improved aquaculture breeding and hatching rates. Probiotics have the potential to treat or prevent COVID-19 by lowering the occurrence and severity of diseases. Lactobacillus casei also interacts with epithelial cells via Toll-like receptors (TLRs) to boost the production of cytokines, which are vital in increasing cell productivity and preventing apoptosis during restoration, promoting survival and proliferation. The preservation of the human GI or lung microbiota may aid in the prevention of COVID-19, since dysbiosis plays an important role in people’s susceptibility to infectious diseases. Most experimental experiments demonstrated that bacteria extracted from processed dairy products belonged to lactic acid bacteria, which are classified as probiotic bacteria. Appropriate sample dilutions were made, and a concentration of just 50 PI-J was dispersed on MRS-agar plates. For 48 h, each plate was kept at 37°C in a static incubator and all the steps were done for performing the tests. Then electrophoresis and pcr is done to confirm the antimicrobial activity.

Various research findings on the importance of probiotics as well as their extraction from processed dairy products are reviewed in this review.