1. Introduction
Nowadays, probiotics have gained major attention due to their diverse range of therapeutic potential. Probiotics are defined as live microorganisms that, when administered in adequate amounts, confer health benefits on the host (WHO/FAO, 2002). The efficacy of probiotic bacteria is greatly influenced by their functional properties, such as antimicrobial activity, persistence in the gastrointestinal tract (GIT) for intestine-targeted probiotics, and immunomodulatory properties [
1,
2] In the gut, probiotics affect balancing and restoration of the gut microbiota, protection against pathogens, immunomodulation, and maintenance of intestinal barrier integrity [
3]. Studies reported that probiotics are frequently used in dietary supplements, food, infant formula formulations, and medical devices [
4,
5]. In addition, probiotics have significant potential as therapeutic options for a variety of diseases, mainly gastrointestinal diseases (including acute infectious diarrhea, antibiotic-associated diarrhea, ulcerative colitis, irritable bowel syndrome, functional gastrointestinal disorders, or necrotizing enterocolitis), as well as extra-intestinal disorders, such as hepatic encephalopathy [
6,
7]. Probiotics not only show the healing effect as live compounds, but they also produce the same pharmacological effect as dead microorganisms, confirming that microbial metabolites present in probiotics have the ability to prevent various types of chronic diseases, including allergy, acute infectious diarrhea, and bowel disease inflammation [
8].
Bifidobacterium and
Lactobacillus microorganisms are most frequently used as probiotics. Interestingly, previous research has been reported that
lactobacillus as a probiotic has potential effect on bacterial infectious diseases caused by
Streptococcus pyogenes and
Streptococcus pneumoniae [
9,
10]. In addition, studies found that the early-stage cold has been inhibited using
Lactobacillus with yogurt [
11]. However, not all probiotics display the same properties; hence, careful selection of specific strains based on their claimed beneficial effects is needed.
Influenza, an infectious disease of birds and mammals, is caused by RNA viruses of the family Orthomyxoviridae (commonly called the influenza viruses). Over the recent pandemic years, between three and five million people have been infected and between 250,000 and 550,000 have died due to the seasonal spread of this deadly influenza virus around the world [
12,
13,
14,
15]. Humans, birds, and pigs are the common host for influenza virus. Based on two specific proteins called hemagglutinin (HA) and neuraminidase (NA), there are different subtypes of influenza viruses [
16]. Influenza virus A H1N1, which affects humans, has some common symptoms, such as fever, pharyngitis, muscle pain, severe headache, include chills, coughing, and weakness [
13,
14]. It also causes pneumonia, which might be fatal in more serious cases, especially for young children and elderly people [
17]. So far, the management authority of influenza disease has been given optimum priority worldwide to control the spread of influenza viruses. In general, people are vaccinated against influenza as a practical and potentially anti-viral therapy. However, considering the limited efficacy of the current vaccination programs [
18] and imposed restrictions on the use of antiviral drugs (those that have severe side effects) [
19], the development of probiotic-based natural anti-viral therapies is in high demand. A few studies have demonstrated that probiotics of some lactic acid bacterial strains were able to protect against infectious diseases [
20,
21] and have the anti-allergic effects on immune diseases in mice [
22,
23] and humans [
24,
25]. Yogurt fermented with
Lactobacillus was shown to reduce the cases of catching cold in the healthy elderly [
11] and to prolong the survival periods of mice with influenza virus infection [
24]. Despite this current situation, there is an alarming demand arising for the development of natural antiviral therapy of probiotic supplements against influenza virus, which might be safe and free from any adversary side effects as the lactic acid bacteria (LAB) and/or probiotics are classified as generally safe (GRAS) by US-FDA. Therefore, the primary objective of this research is to isolate, identify, and characterize the novel probiotic strain
L. plantarum YML015 and to evaluate its potent antiviral efficacy against influenza virus A H1N1 in vitro and in ovo.
4. Discussion
Nowadays, the use of probiotics is in high demand due to their high immunostimulant potency. For example, the probiotic strain
Lactobacillus plantarum KLAB21 from Korean kimchi has significant antimutagenic characteristics [
45,
46]. There is a huge trend among researchers to explore and study different probiotics or Lactic Acid Bacteria (LAB) from Korean fermented food products. Studies have also observed their chemical nature, along with antimicrobial potential against various food spoilage and food-born pathogens, including molds and fungi, for their possible use as food preservatives [
29]. In this current study, a total of 1200 LAB strains were isolated from different kinds of Kimchi samples, which were initially screened for their antiviral efficacy. Among them, one of the strains designated as
L. plantarum YML015 exhibited remarkable antiviral activity against IFVA H1N1. The 16 s rRNA sequencing confirmed molecular characterization of the YML015 isolate as
L. plantarum YML015. The selected strain was also further identified based on the biochemical characterization utilizing carbohydrate interpretation assay (API 50CHL kit). A standard method of carbohydrate utilization according to API web software showed that
L. plantarum YML015 used approximately 24 carbohydrates among 50 carbohydrates (
Table 1), thus, was characterized as Gram-positive and
lactobacilli.
In our study, the probiotic nature of
L. plantarum YML015 was confirmed based on the susceptibility test result. However, the European Food Safety Association (EFSA) has already published a new protocol for foods and feeds purposes, which might be helpful to evaluate probiotic strains as a food grade probiotic [
47]. Moreover, “Qualified Presumption of Safety” (QPS) also described the importance and procedure of determination of antibiotic resistance for selected probiotic strains. We experienced that the probiotic strain
L. plantarum YML015 was very sensitive to clinically essential antibiotics such as ampicillin, gentamycin, kanamycin, streptomycin, erythromycin, clindamycin, and tetracycline. However, phenotypic antibiotic resistance testing in LAB requires standardization as it can be subjected to species specificity but is also dependent on the method and the media used [
48]. Eventually,
L. plantarum YML015 was found to cause γ-hemolysis (non-hemolysis). Results proved again that
L. plantarum YML015 might be a potential probiotic with non-hemolytic characteristics. It is also a common phenomenon of non-hemolytic LAB isolates from food sources [
49,
50], except some strains of
Enterococcus faecalis. Furthermore,
Table 2 describes temperature stability of the cell-free supernatant of
L. plantarum YML015. Interestingly, at optimum temperature treatment of 121 °C for 15 min, antiviral activity of CFU of
L. plantarum YML015 was the same up to 96 h of activity observation. In addition to the MTT cell viability study, it has been significantly demonstrated that
L. plantarum YML015 has optimum cell viability percentage with no cell damage or cytotoxic effect (
Figure 4). The result supported that, as a probiotic,
Lactobacillus plantarum YML015 is highly safe for human and animal consumption. The study also found that heat-killed bacteria, especially heat-treated probiotic strains, showed immunomodulating effects and had antagonizing properties against pathogens [
51]. Hence, it was confirmed that
L. plantarum YML015, as a strong probiotic, could be used as a heat-stable and effective agent against the H1N1 virus.
Viral invasion causes structural damage or change in the host cells called cytopathic effect (CPE). Two kinds of effects might occur due to CPE, such as the infecting virus leading to lysis of the host cell or because of the inability to reproduce new cells (cell death). It is also called cytopathogenic when morphological changes in the host cell are caused by virus. Moreover, [
42,
52] described that probiotic
Lactobacilli strains exhibited antiviral effect against influenza virus as experienced with the cytopathic reduction assay. In our experiment, we successfully confirmed that newly invented
L. plantarum YML015 has significant potential to inhibit the cytopathic effect caused by IFVA H1N1. It was ensured that the cell-free supernatant (1-fold and 10-fold) of
L. plantarum YML015 completely suppressed the cytopathic effect of IFVA H1N1 on a monolayer of the MDCK cell line as compared with the negative control as described in
Figure 5.
The hemagglutination inhibition assay is a popular screening assay. It allows rapid detection of antiviral activity of any kinds of sample against influenza virus [
33,
53]. This assay was standardized on the basis of the ability of viral HA protein to bind and to coagulate/agglutinate human or chicken red blood cells (RBC) [
39]. Our findings are aligned with the previous observations, and it was ascertained in our research work that CFS of
L. plantarum YML015 exhibited most effective potential in the hemagglutination inhibition activity assay (
Table 5). This strongly suggests that probiotic dietary uptake of
L. plantarum YML015 could be beneficial for direct inhibition of influenza virus A H1N1 infection [
42].
In addition, this research also assessed the in ovo potential of the probiotic strain L. plantarum YML015 to reconfirm the antiviral efficacy on influenza A virus H1N1 and was expressed as a percentage of survival from the total number of test samples. Among all treated samples, including the negative control, only heat-killed CFS (10-fold) and CFS (10-fold) showed highest antiviral activity compared to other tested samples, and the survival rate was 100%. However, inoculation of eggs with standard treatment (Tamiflu) showed a 50% survival rate, suggesting that as a probiotic, L. plantarum YML015 could be a good source of natural anti-influenza drugs.