Probiotic Properties of Lactobacillus helveticus and Lactobacillus plantarum Isolated from Traditional Pakistani Yoghurt

Probiotic bacteria are of utmost importance owing to their extensive utilisation in dairy products and in the prevention of various intestinal diseases. The objective of this study was to assess the probiotic properties of bacteriocin-producing isolates of Lactobacillus helveticus and Lactobacillus plantarum isolated from traditional Pakistani yoghurt. In this study, ten bacteriocin-producing isolates were selected to screen for the probiotic property. The isolates showed resistance to acidic pH (6-6.5), bile salt (0.01-1%), and 1-7% NaCl salt and showed good growth at acidic pH and antibacterial activity against ten different foodborne pathogens. Interestingly, these isolates were proved to be effective against Actinobacter baumannii but least effective against Klebsiella pneumoniae and Pseudomonas aeruginosa. A few isolates were found to be resistant to some antibiotics like vancomycim, gentamycin, erythromycin, streptomycin, and clindamycin. Our results provide strong evidence in favour of traditional Pakistani yoghurts as a potential source of bacteriocin-producing bacteria with an added benefit of the probiotic property. Specifically, LBh5 was considered a good probiotic isolate as compared to other isolates used in the study. Further extensive research should be done on isolation and characterisation of probiotic isolates from local fermented foods, and then, these isolates should be used in the development of probiotic enriched food supplements in Pakistan.


Introduction
Probiotic is a well-known term used for "live microorganisms" when added as supplements in food, which provide many health benefits. In the food industry, lactic acid bacteria (LAB) are the most promising group of bacteria that have been consumed fearlessly as they are generally regarded as safe (GRAS) [1]. In fact, most of them are natural inhabitants of dairy products like cheese and yoghurt and considered native microflora in dairy products [2,3]. They are present naturally or added purposely to food products for enhancing their flavour and aroma and for the maintenance of human gut microflora. Searching fermented food items for probiotics has been a very practical and convenient approach for preservation as it does not invite objections against chemical additives being a part of the intake [4]. The present study includes Lactobacillus, the probiotic genus popular for many medical applications such as decreasing enteric infections, intestinal tumors, and cholesterol levels, folate production, treating cardiovascular diseases, metabolic disorders, lactose intolerance, and boosting the immune system [5][6][7][8][9]. Moreover, such bacteria release anticarcinogenic and antimicrobial substances in addition to organic acids such as lactic acid, benzoic acid, and acetic acid [5,10]. The antimicrobial peptides known as bacteriocins are also defining characteristics of these bacteria, which efficiently inhibit the growth of certain foodborne pathogens and spoilage bacteria [11]. Among lactobacilli, Lactobacillus plantarum and Lactobacillus helveticus are two probiotics commonly used as a starter culture in several fermentations, especially in the fermentation of dairy products [4]. Lactobacillus helveticus shows profound proteolytic activity and is also reported as a good inhibitor of angiotensin-converting enzyme, which has a role in alleviating hypertension. Lactobacillus plantarum is an indigenous gut inhabitant and therefore is considered more compatible with the gut environment [12,13].
The antimicrobial ability maintained by a probiotic is attributed to the production of organic acids (acetic acid and lactic acid), protein metabolites, hydrogen peroxide, cyclic dipeptides, enzymatic effects, and certain antimicrobial peptide-bacteriocins [14,15]. Lately, isolation and characterisation of bacteriocins are of interest to microbiologists as they could be a better candidate to replace antibiotics and synthetic preservatives [4]. In the past decades, many Lactobacillus species were reported for their probiotic potential and exhibited antagonistic activity against various microbes, but very few studies have been done on bacteriocinproducing potential probiotic strains isolated from a dairy product.
Isolating potential probiotic strains has been practiced for centuries and is still of interest because of its industrial and medicinal value [16]. Probiotic strains must have the ability to tolerate the stress environment contained in the gastrointestinal system. Their viability in harsh conditions like the presence of bile, gastric juices, and NaCl, low pH in the stomach, adhesion to the intestinal lining, and antibiotics contributes to their efficacy [8,17]. Their resistance to environmental stress and their bacteriocin producing ability would aid the food industry in establishing safe preservation strategies. In Pakistan, yoghurt is a famous dairy product obtained by fermentation of milk. It is consumed daily by a large population as it possesses nutritional value; plus, it is a significant source of beneficial bacteria [17]. Yoghurt from different countries has been investigated for the isolation of potential probiotics. Literature shows that a distinctive microbiome has also been reported from yoghurt of different cities of Pakistan, such as Lahore, Faisalabad, Karachi, Islamabad, and Peshawar. These isolates include Lactobacillus bulgaricus, Lactobacillus casei, Lactobacillus acidophilus, Lac-tobacillus salivarius, and Leuconostoc mesenteroides CYG362 [18,19] from Lahore; L. acidophilus, Lactobacillus delbrueckii subsp. bulgaricus, Lactobacillus paracasei subsp. paracasei, L. bulgaricus, L. delbrueckii, L. plantarum, Lactobacillus fermentum, Lactobacillus rhamnosus, and Streptococcus thermophilus from Faisalabad [20][21][22][23][24]; L. bulgaricus from Islamabad [25]; Pediococcus pentosaccus, L. delbrueckii, L. plantarum, L. helveticus, and Pediococcus acidilactici from Peshawar; and Lactobacillus curvatus KIBGE-IB44 from Karachi [26,27]. These microbes are also included in commercial probiotic yoghurt in Pakistan [28]. In Pakistan, L. helveticus and L. plantarum are rarely isolated from yoghurt as compared to other species; thus, their probiotic potential is barely determined.
In our previous study [29], we isolated bacteriocin from two lactobacilli, L. plantarum and L. helveticus, which effectively inhibited foodborne pathogens. In this study, we extended our research and evaluated the probiotic potential of bacteriocin-producing species, as very few bacteriocin producing probiotic species were described earlier. This study provides potent probiotics that could be used in food preservation and storage strategies to prevent certain spoilage bacteria and thus reduces the economic loss experienced by the food industry.

Contributions.
Our contributions in this study are as follows: (i) In an earlier study, an analysis of the bacteriocin properties of Lactobacillus species (isolated from traditional yoghurt) was done. Among different isolates, Lactobacillus species L. helveticus and L. plantarum were involved in the production of bacteriocins. In the current study, these two species were evaluated for probiotic properties. The comparative analysis of probiotic properties on both species showed that L. helveticus was the most suitable probiotic species as compared to L. plantarum against foodborne pathogens (ii) Among these two species, L. helveticus was seldom found in dairy products. It was the first time that these bacteriocin-producing isolates have been isolated from a yoghurt sample from Peshawar KPK while L. plantarum has been isolated a few times from dairy products. These isolates could further be subjected to various in vivo approaches to find the actual target of these isolates. Furthermore, these isolates could be assessed for the production of the unusual proteolytic spectrum and other inhibitors (iii) The remarkable probiotic properties of these two bacteriocin-producing species and the comparison of L. helveticus and L. plantarum make this study distinctive and interesting for readers  [30]. Bacterial growth was monitored spectrophotometrically at 600 nm (A 600 ) at 0, 2, 4, 6, 8, 10, 12, and 24 hrs. The measurements were carried out using an Agilent Cary 5000 Ultraviolet-to-Visibleto-Near-Infrared (UV-Vis-NIR) spectrophotometer (Agilent Technologies, Petaling Jaya, Malaysia) [31].

Evaluation of Probiotic Properties.
To evaluate the probiotic potential of bacteriocin-producing isolates, the following properties were considered: tolerance to low pH, bile salts , NaCl, antibiotic susceptibility, antimicrobial activity, and response to gastroduodenal stimuli.
2.4.1. Low pH Tolerance. A 1 mL bacterial culture was inoculated into eight tubes, each containing 9 mL MRS broth adjusted to varying pH ranges from 1 to 8. After 4, 6, 12, and 24 hours of incubation at 37°C, the growth rate was calculated by considering the optical density (OD) values measured at 600 nm. Uninoculated media was taken as a negative control.

NaCl Tolerance.
Isolates were drawn in MRS broth having different NaCl concentrations ranging from 1 to 7%, incubated at 37°C for 4, 6, 12, and 24 hrs at 37°C. Growth was determined by measuring the optical density of the broth at 600 nm. A tube without NaCl was run as a negative control.

Antibiotic Susceptibility
Test. The agar well diffusion method was used to test antibiotic susceptibility against fre-quently used antibiotics. The antibiotics which were used in this study against all ten isolates with the same concentrations are ampicillin (Amp) (2 mg/L), gentamycin (Gen) (16 mg/L), erythromycin (Ery) (1 mg/L), and clindamycin (CLI) (4 mg/L). The concentration of kanamycin used for the L. plantarum isolates was 64 mg/L and for the L. helveticus isolates 16 mg/L. The two antibiotics tetracycline (Tet) at 32 mg/L and chloramphenicol (CL) at 8 mg/L were only tested against L. plantarum isolates. The two antibiotics streptomycin (SM) at 16 mg/L and vancomycin (Van) at 2 mg/L were only tested against L. helveticus isolates. The stock solution of the antibiotics was made in distilled water according to guidelines from the Clinical and Laboratory Standards Institute (CLSI) and the European Food Safety Authority (EFSA) [32,33]. Distilled water was run as a control.   In the study of Cho et al. [37], the isolates of L. plantarum reached stationary phase after 10 hours. Balamurugan et al. [38] also reported that L. helveticus showed an elevated growth curve at around 16 hours in MRS broth, which was similar to our results as shown in Tables S1 and S2.

Evaluation of Probiotic Properties
3.3.1. pH Tolerance. A low pH tolerance test is essential to predict the survival of the isolates in the stomach environment. It was observed that L. plantarum isolates showed negligible growth up to pH 4, but at pH 5-6, an increase in growth rate was observed. The decline in the growth rate of L. plantarum isolates occurred at pH above 6; the growth rate declines drastically. Chakraborty and Bhowal [39] reported that L. plantarum showed maximum growth at pH 5-6, which was quite similar to our findings. Lactobacillus helveticus isolates showed tolerance up to pH 8. Guetouache and Guessas [40] had also reported that L. helveticus showed tolerance at alkaline pH. Most of the isolates showed a decrease in the tolerance towards pH up to 6 hours of incubation; after that, they showed a sharp decline in their growth rate, as shown in Figure 2. Lactobacillus helveticus isolates showed better tolerance to high pH as compared to L. plantarum.

BioMed Research International
The isolate LBh5 showed higher tolerance against higher pH (up to pH 8) as well as a lower pH as compared to other isolates used in this study. LBh1 showed the most significant results, among all other isolates. The statistical analysis is as given in Tables S3 and S4. 3.3.2. NaCl Tolerance. Tolerance to a high concentration of NaCl is necessary for a probiotic to be effective in the human gut. NaCl tolerance was considered to be an important parameter because NaCl concentration is a scale to measure how much bacteria are able to tolerate toxic and osmotic shock. For both the Lactobacillus spp., optical densities decrease gradually with an increase in NaCl concentrations, as shown in Figure 3. Lactobacillus plantarum isolates comparatively showed higher OD values than L. helveticus isolates. Both lactobacilli showed growth at 1-4% NaCl concentration, while at a higher concentration of NaCl, from 6 to 7%, bacterial growth was decreased. Balamurugan et al. [38] also reported that L. helveticus showed tolerance to NaCl concentration from 1 to 7%, and Chowdhury et al. [41] observed that L. plantarum was able to tolerate NaCl concentration from 1 to 9%: both of these studies matched our findings. The isolates LBh5 showed the most substantial tolerance to NaCl concentration as compared to other isolates of L. helveticus. In L. plantarum isolates, LBp3 showed the most favourable results among the others, as shown in Figure 3; it was concluded that LBh5 showed the most positive results as compared to all isolates of L. plantarum and L. helveticus used in this study. The results were statistically analysed as shown in Tables S5 and S6. 3.3.3. Bile Salt Tolerance. Bile salt tolerance is an essential factor as it determines the survival of probiotics in the intestine. The bile salts present in the intestinal tract disrupt the cell membrane of bacteria entering the stomach. Probiotics have the ability to tolerate 0.05 to 0.3% of bile. The results calculated showed that the maximum growth of isolates was observed in the presence of bile salts up to 0.3%, as shown in Figure 4. The isolates, however, were less tolerant of the higher concentrations, i.e., 0.6% and 1%. Chowdhury et al. [41] observed that Lactobacillus subsp. isolated from yoghurt tolerated around 0.3% of bile concentrations. Lactobacillus helveticus isolates showed tolerance up to 0.3%, and maximum tolerance was observed at 0.1%. Barua et al. [42] and Rong et al. [43] observed that Lactobacillus spp. showed maximum growth at 0.1%, and maximum tolerance was shown at 0.3% concentration, which correlated to this study. Similar findings were reported in a work done by Baick and Kim [44]. The isolates LBp1 and LBp2 were able to withstand a higher bile salt concentration up to 1% as compared to other isolates of L. helveticus and L. plantarum. On the contrary, the findings of Succi et al. [45] showed the survival of lactobacillus bacteria at high bile concentrations up to 1%. The isolate LBh4 showed maximum tolerance, i.e., up to 0.3% as compared to other isolates of L. helveticus. It was concluded that L. plantarum and L. helveticus isolates can be considered

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BioMed Research International to be suitable probiotic isolates as they tolerated 0.3% of bile concentration, which is typical of healthy men [9]. The statistical analysis of the result is shown in Tables S7 and S8. 3.3.4. Resistance to Antibiotics. Potential probiotic bacteria show a natural resistance to antibiotics. All L. plantarum isolates exhibited resistance towards clindamycin. Anas et al. [46] reported similar results in their study. The L. plantarum isolates showed sensitivity against ampicillin, gentamycin, and other antibiotics, and among all isolates, LBp1 showed the maximum zone of inhibition against kanamycin (38.1 mm), as shown in Figure S1. The isolates LBp3 and LBp5 showed resistance against gentamycin, kanamycin, erythromycin, clindamycin, and tetracycline. It was concluded that among these five isolates, LBp3 and LBp5 were resistant against most of the drugs. Similar observations were reported by Somashekaraiah et al. [47]. LBh3 and Lbh5 showed resistance against vancomycin, gentamycin, streptomycin, erythromycin, and clindamycin, as shown in Figure 5. All the isolates of L. helveticus showed sensitivity towards kanamycin and ampicillin. In the case of clindamycin, all the four isolates of L. helveticus showed resistance except LBh2 isolate, which was sensitive to this drug. The isolate LBh2 was sensitive to gentamycin with a maximum zone of inhibition of 25.7 mm. The antibiotic susceptibility tests showed that L. helveticus isolates had better probiotic properties as compared to L. plantarum isolates. Most of the L. helveticus isolates were found to be sensitive to all antibiotics used in this study as compared to L. plantarum isolates. Resistance to various antibiotics would help the isolates settle in gut microflora for longer; therefore, may alleviate gastrointestinal tract-related side effects due to antibiotics. Tables S9 and S10 show the statistical analysis of the results.
3.3.5. Antibacterial Activity. The antibacterial activity of L. plantarum and L. helveticus was measured against foodborne pathogens. All L. plantarum and L. helveticus isolates showed a maximum zone of inhibition against St. aureus (22.9 mm and 19.5 mm, respectively). In contrast, minimum zones were found against the gram-positive bacterium S. pyogenes (6.8 mm and 4.8 mm), as shown in Figure 6 and Figure S2. Al-Madboly and Abdullah [48] Figure 6: Antibacterial activity of different L. plantarum and L. helveticus isolates against foodborne pathogens. The tree is a dendrogram, which shows the hierarchy (similarity) between isolates and their activity against the different foodborne pathogens. The graph was plotted using R program.
14 BioMed Research International zone of inhibition against K. pneumoniae (5 mm), and L. helveticus isolates showed a minimum zone of inhibition against P. aeruginosa (5.6 mm). The isolates LBh5 showed significant results against gram-positive bacteria, while LBh3 isolates showed the most promising results against gram-negative bacteria as compared to other L. helveticus isolates. Gupta et al. [49] observed the same results in the case of L. helveticus antibacterial activity. The isolate LBp5 showed the most promising results against both grampositive and gram-negative bacteria in comparison to all other isolates used in this study. Tables S11 and S12 present detailed results of the Pearson correlations.
3.3.6. Response to Stomach-Duodenal Stimulus. The response of lactic acid bacteria isolates to stomach-duodenal stimulus is shown in Figure 7. These results indicated that there was a resistance of most isolates to adverse conditions imposed by the composition of this medium. All the tested isolates were resistant to pH 3 after 2 hrs of incubation. Similar results were reported by Vizoso Pinto et al. [34], although their isolates showed resistance after 1 hour of incubation.
3.3.7. Arginine Hydrolysis Test. Both the isolates were shown to be arginine-positive as on a white background they displayed a bright orange colour.

Conclusion
Bacteriocin-producing lactic acid bacteria were successfully isolated from traditional Pakistani yoghurt. The experimental results showed that both L. plantarum and L. helveticus isolates were able to tolerate low pH, high bile salt, and NaCl concentrations. They also showed significant antimicrobial activity against test foodborne microorganisms and were resistant to many antibiotics. Thus, our isolates proved that they bear probiotic potential and could be used in different food items as probiotics. The L. helveticus isolates (LBh5) showed good probiotic properties as compared to other isolates investigated in this study. A further study on these isolates is needed to explore more about their role in increasing the shelf life of food or preventing or treating any gastrointestinal infections. The data was authenticated by replication. The Pearson correlation was applied which showed most of the results with significant values.

Limitations of the Study.
Despite the promising results and uniqueness of our study, these isolates should be further studied to prove their importance in the dairy industry. It would be valuable to test the following characteristics: (i) molecular identification of isolates; (ii) adhesion to mucosal surfaces; (iii) clinical studies for human health; and (iv) technological properties (strain stability, viability into products, and bacteriophage resistance).

Data Availability
The authors will provide the data if needed.

Conflicts of Interest
The authors report no conflict of interest.