Greener Journal of Biological Sciences

Vol. 9(2), pp. 22-28, 2019

ISSN: 2276-7762

Copyright ©2019, the copyright of this article is retained by the author(s)

DOI Link: http://doi.org/10.15580/GJBS.2019.2.071719137

http://gjournals.org/GJBS

 

Description: C:\Users\user\Pictures\Journal Logos\GJBS Logo.jpg

 

 

 

 

Antagonistic effect of developed probiotic yoghurt against some selected food-borne pathogens during cold storage

 

 

* 1Aforijiku, S., 2Onilude, A. A. and 3Wakil, S.M.

 

 

1 Department of Microbiology, University of Ibadan, Ibadan, Oyo State, Nigeria.

2 Department of Microbiology, University of Ibadan, Ibadan, Oyo State, Nigeria.

3 Department of Microbiology, University of Ibadan, Ibadan, Oyo State, Nigeria.

 

 

 

 

ARTICLE INFO

ABSTRACT

 

Article No.: 071719137

Type: Research

DOI: 10.15580/GJBS.2019.2.071719137

 

 

This study was carried out to investigate the antagonistic effect of probiotic yoghurt against some selected food-borne pathogens during cold storage. Probiotic yoghurt was produced from processed cow milk using controlled fermentation, and later inoculated with selected food-borne pathogens at inoculum level of 105 CFU/mL. The antagonistic effect of the probiotic yoghurt against the food-borne pathogens during cold storage (4oC) for 2 days was studied using standard methods. The results obtained demonstrated that the probiotic yoghurt inhibited growth of food-borne pathogens including Salmonella typhimurium ATCC13311, Proteus mirabilis ATCC25933, and Escherichia coli ATCC2592 in the yoghurt within 24 hr. Their counts decreased from 105-102 CFU/mL, with pH and probiotic counts ranging between 4.48-4.35, and 106-108 CFU/mL, respectively. The probiotic bacteria have the ability to suppress the growth of pathogen like E. coli in yoghurt. This inhibitory effect may be due to low pH of yoghurt. The probiotic cultures in the yoghurt can be used as biopreservatives in food and pharmaceutical industries.

 

Accepted:  19/07/2019

Published: 01/08/2019

 

 

*Corresponding Author

Aforijiku, S

E-mail: dayophd@ yahoo.com

Phone: 08028037495

 

Keywords: Food-borne pathogens; probiotic yoghurt; antagonistic effect; inoculum level

 

 

 

 

 

 

INTRODUCTION

 

Lactic acid bacteria are Gram positive rods or cocci, which have the ability to produce lactic acid. Lactic acid bacteria (LAB) could be used successfully with limited or no negative effects, to control challenging problems associated with some enterics.The substances produced by the LAB, are kept in the foods which could help to inhibit pathogens (Brant and Todd, 2014; Mohammed et al., 2016).  The antimicrobial potential of LAB can be due to ability in producing substances like lactic acid which has the tendency to suppress pathogenic microbes (Brant and Todd, 2014; Tribe et al., 2014; Nikolic et al., 2008).  In addition to production of organic acids, the pH reduction of the products or medium can have antagonistic effect on pathogens.

Some studies have reported that strains of Salmonella typhimurium, and other pathogens were suppressed by substances produced by LAB ((Brant and Todd, 2014; Tribe et al., 2014; Evariste et al., 2017; Gopalakrishnan, 2018). Therefore, the probiotic LAB make the environment unfavourable for pathogens to thrive during the manufacture of probiotic yoghurt, hence limiting the viability of the pathogens (Mohammed et al., 2016;  Ting and Xialian, 2019). Moreover, milk and milk products are usually affected by pathogen like E. coli and Salmonella typhimurium, since some strains can survive acidic conditions (Bibbal et al., 2014; Rebello et al., 2014; CDC, 2016; Evariste et al., 2017). However, there is little attention on the survival of these pathogens in yoghurt. Therefore, there is a need to assess the antagonistic effect of probiotic yoghurt against food-borne pathogens during cold storage.

 

 

MATERIALS & METHODS

 

Collection of samples

 

Raw milk from white Fulani cow was purchased from Dairy and Research Farm, University of Ibadan, Ibadan, Nigeria. It was brought into Physiology Laboratory, at the Department of Microbiology in sterile bottles for production of yoghurt.

 

Collection of Indicator organisms

 

Indicator organisms such as Salmonella typhimurium ATCC13311, Proteus mirabilis ATCC25933, and Escherichia coli ATCC25922 were obtained from the culture collection unit of Federal Institute of Industrial Research, Oshodi (FIIRO).

 

Preparation of inoculum size of pathogens

 

Each strain of Salmonella typhimurium ATCC13311, Proteus mirabilis ATCC25933 and Escherichia coli ATCC25922 were inoculated in 10 mL tryptic soya broth containing 0.6 % yeast extract, and incubated at 370C for 24 hr. The serial dilutions was made, and the inoculation level was determined by direct plating on specific media of the pathogens from serial dilution of broth.  Inoculum size of 105 CFU/mL was used (ISO, 2003).

 

Probiotic  cultures

 

Potential probiotic starters such as Lactobacillus plantarumN24, Lactobacillus plantarumN17, Lactobacillus brevis N10, and Lactobacillus caseiN1 isolated from nono samples were used to produce yoghurt.

 

Antagonistic effect of probiotic yoghurt against food-borne pathogens

 

Yoghurt was prepared in the laboratory using method described by Rahmann et al. (1999). For each yoghurt samples, 100 mL of the cow milk was heated to 85oC for 30 minutes, and then cooled immediately in an ice bath to temperature of 370C. This was then, inoculated with 106 CFU/mL probiotic starters, and incubated at 42oC for 4 hr. After yoghurt formation, yoghurt was inoculated with the pathogens at inoculation size of 105 CFU/mL. The yoghurt without inoculation of pathogens (control), and the inoculated yoghurt were stored in the refrigerator. The yoghurt samples were examined microbiologically for pathogens count during 2 days of storage, using pour plate technique in Petri dishes. The viability of probiotic cultures and pathogenic organisms was done every 24 hr during 2 days of storage according to  methods of 1SO (2003) and (1SO, 2004), respectively. One mL of appropriate dilutions of yoghurt samples were plated on Mac Conkey agar (for Proteus), Eosin methylene blue agar (for E. coli), Salmonella Shigella agar for Salmonella, and MRS agar for lactic acid bacteria, and incubated at 37oC for 48 hrs.  Colony forming unit were then estimated. The pH was determined using a pH meter, following the manufacturer’s instructions (APHA, 2004). The experiments were done in duplicates.

 

Statistical analysis

 

The values for each parameters were calculated and presented as means of duplicates. Data was analysed using Analysis of Variance (ANOVA) with Duncan Multiple Range Test for significance at P≤0.05. Standard deviation was not shown. Data were also presented in tables.

 

 

RESULTS AND DISCUSSION

 

The codes of prepared probiotic yoghurt with pathogens and without pathogens are shown in Table 1. Table 2 showed the effect of probiotics against Proteus mirabilisATCC 25933, during storage period at 4oC for 2 days, when the initial inoculum size of the pathogen was 105 CFU/mL.  At first day of storage, the count of Proteus mirabilis reduced from 105 to103 and 102 CFU/mL, and their counts were significantly different (P≤0.05) in probiotic yoghurt samples, when pH ranged from 4.40-4.45. By the second day, the inoculated Proteus mirabilis was not found in the probiotic yoghurt, when probiotic cultures count was between 106 to 108 CFU/mL, and pH ranged between 4.34- 4.42.

However, similar studies also demonstrated that the pathogen was not detected after 48 hr of storage period as reported by Bachrouri et al. (2006). This could be as a result of increased probiotic cultures count, pH and probiotic strain used to produce yoghurt. Our findings are in accordance with the work of Ting and Xialian. (2019) in terms of inhibition at 24 hr of cold storage. They studied antagonistic effect on Proteus mirabilis, and some spoilage organisms in yoghurt fermented with probiotic starters, these pathogens were  inhibited completely within 24-48 hr. The inhibition of pathogens could be as a result of probiotic starters strain used to produce yoghurt (Wang et al., 2004).

Table 3 showed the antagonistic effect of probiotic cultures against Salmonella typhimurium ATCC13311 during the cold storage (4oC) for 2 days with the initial inoculum size of Salmonella typhmurium ATCC13311 at 105CFU/mL. At first day of storage, the initial count of the pathogen decreased from 105 -102 CFU/mL, but not found in sample YN24-N17, when the pH was 4.38. At second day, Salmonella typhimurium disappeared in sampleYN17, with pH4.37, and probiotic count (2.0x107 CFU/mL), which increased to 108 CFU/mL.


 

 

Table 1: Codes of prepared probiotic yoghurt

Samples

Prepared probiotic yoghurt (inoculated with pathogens)

Prepared probiotic yoghurt (without pathogens)

1

YN24

yn24

2

YN17

yn17

3

YN10

yn10

4

YN1

yn1

5

YN24-N17

yn24-n17

6

YN24-N10

yn24-n10

7

YN24-N1

yn24-n1

8

YN17-N10

yn17-n10

9

YN17-N1

yn17-n1`

10

YN10-N1

yn10-n1

  *Samples with Capital letters codes (prepared probiotic yoghurt inoculated with pathogens)

                            *Samples with small letters codes (prepared probiotic yoghurt without pathogens)

 


The experiment was done in duplicates

 

Keys:

 

1-Yoghurt made from cow milk and Lactobacillus plantarumN24

2-Yoghurt made from cow milk and Lactobacillus plantarumN17

3 -Yoghurt made from milk and Lactobacillus brevisN10

4 -Yoghurt made from cow milk and Lactobacillus caseiN1

5-Yoghurt made from cow milk and Lactobacillus plantarumN24 &Lactobacillus plantarumN17

6-Yoghurt made from cow milk and Lactobacillus plantarumN24&Lactobacillus brevisN10

7-Yoghurt made from cow milk and Lactobacillus plantarumN24 &Lactobacillus caseiN1

8-Yoghurt made from cow milk and Lactobacillus plantarumN24 &Lactobacillus brevisN10

9-Yoghurt made from cow milk and Lactobacillus plantarumN17 &Lactobacillus caseiN1

10-Yoghurt made from cow milk and Lactobacillus brevisN10 &Lactobacillus caseiN1,


 

 

Table 2: Antagonistic effect of developed probiotic yoghurt against of Proteus mirabilis ATCC25933 stored under cold storage (4oC)

 

Storage time(days)

   1

Bacterial

Count(CFU/mL)

 

 

 

2

 

Samples

Codes

pH

PMC

pH

PMC

PCC

YN24

4.42b

2.1x103a

4.40a

-

5.0 x106c

yn24*

4.40c

-

4.39ab

-

1.2x107b

YN17

4.43ab

2.3x103a

4.42a

-

6.0 x106b

yn17

4.40c

-

4.40a

-

6.9x106b

YN10

4.45a

3.5x103a

4.39ab

-

1.2x107b

yn10

4.43ab

-

4.39ab

-

6.4x106b

YN1

4.44a

1.7x103a

4.40a

-

5.8x106b

yn1

4.41c

-

4.38ab

-

6.7x106b

YN24-N17

4.41c

1.8x102b

4.38ab

-

2.0x107b

yn24-n17

4.40c

-

4.38ab

-

3.1x107b

YN24-N10

4.40c

1.2x102b

4.36b

-

1.8x107b

yn24-n10

4.39c

-

4.36b

-

3.7x107b

YN24-N1

4.42b

3.0x102ab

4.38ab

-

1.5x107b

yn24-n1

4.41c

-

4.36b

-

2.7x107b

YN17-N10

4.40c

1.2x102b

4.35b

-

1.3x107b

yn17-n10

4.40c

-

4.37ab

-

1.4x108a

YN17-N1

4.42b

1.2x102b

4.39ab

-

2.0x107b

yn17-n1

4.43ab

-

4.37ab

-

3.5x107b

YN10-N1

4.40c

1.3x102b

4.34b

-

1.8x107b

yn10-n1

4.42b

-

4.35b

-

2.8x107b

Means with the same alphabets within a column are not significantly different at P≤0.05 using Duncan Multiple Range Test (DMRT). Data collected were represented as “Means of duplicates. Standard Deviation (SD)”not shown., - = Not viable, initial and inoculum size of Proteus mirabilis= 105 CFU/mL, PMC =Proteus mirabilis count, PCC = Probiotic cultures count, inoculum size of starters=106 CFU/mL, PCC for the first day= all were 106 CFU/mL.

*All small letters (yoghurt without pathogen) Samples with capital letters (yoghurt inoculated with Proteus mirabilisATCC25933)

 


 

 

This is in accordance with the work of Al-Delanmy and Hamamdeh (2013) that reported the inhibition of Salmonella typhimurium during 48 hr of cold storage. The reasons for suppression of pathogen could be low pH, and inability to compete with the probiotic cultures for nutrients (Wang et al., 2004; Tsegaye and Ashenafi, 2005; Donkor et al., 2006; Gopalakrishnan, 2018, Nassib et al., 2006, Ting and Xialian, 2019).  Probiotics LAB could prevent growth of pathogens due to low pH. The variation observed by various scientists might be due to difference in survival of strain to lowered pH and temperature, type and strain of starter cultures being used, inoculum size of starters, and the pathogens (Tsegaye and Ashenafi, 2005).The probiotic bacteria have the ability to prevent the growth of pathogens due to low pH initiated by LAB. The fermentation time and temperature, type of probiotic organisms, increased probiotic cultures count, acid tolerance, and the strain of the pathogenic organisms could play important role on the survival of food pathogens in yoghurt. Donkor et al. (2006) concluded that the ability of probiotic to survive in yoghurt was  strain dependent.


 

 

Table 3:  Antagonistic effect of developed probiotic yoghurt against Salmonella typhimuriumATCC13311  during cold storage(4oC).

Storage time (days)                        1

Bacterial count (CFU/mL)

 

 

 

   2

 

 

 

Samples

Codes

pH

STC

pH

STC

PCC

YN24

4.40ab

3.3x103a

4.39ab

-

1.0 x107b

n24 *

4.40a

-

4.38ab                

-

1.9x107b

YN17

4.42a

2.3x103a

4.37b

-

2.0 x107b

yn17

4.40a

-

4.37b

-

2.5x107b

YN10

4.41a

1.9x103a

4.38ab

-

3.0x107b

yn10

4.41a

-

4.37ab

-

3.6x107ab

YN1

4.41a

1.4x103a

4.40a

-

3.3x107b

yn1

4.40a

-

4.38ab

-

3.8x107ab

YN24-N17

4.38ab

-

4.36b

-

1.1x108a

Yn24-n17

4.36b

-

4.36b

-

1.9x108a

Y24-N10

4.36b

-

4.35b

-

1.3x108a

yn24-n10

4.35b

-

4.35b

-

1.6x108a

YN24-N1

4.40a

1.2x102b

4.39ab

-

2.5x107b

yn24-n1

4.39ab

-

4.37b

-

2.7x107b

YN17-N10

4.37ab

-

4.36b

-

1.1x108a

yn17-n10

4.36b

-

4.36b

-

2.2x108a

YN17-N1

4.41a

1.5x102b

4.39ab

-

3.2x107b

yn17-n1

4.40a

-

4.38ab

-

3.7x107ab

YN10-N1

4.37ab

-

4.35b

-

1.4x108a

yn10-n1

4.36b

-

4.35b

-

1.9x108a

Means with the same alphabets within a column are not significantly different at P≤0.05 using Duncan Multiple Range Test (DMRT). Data collected were represented as “Means of duplicates. Standard Deviation (SD)”not shown.

- = Not viable, initial and inoculum size of Salmonella typhimurium= 105 CFU/mL, STC =Salmonella typhimurium count, PCC = Probiotic cultures count, inoculum size of Probiotic starters and PCC at the first day=106 CFU/mL,

*All small letters (yoghurt without pathogens),

*Samples with capital letters (yoghurt inoculated with Salmonella typhimuriumATCC13311)

 

 


Table 4 showed that E coli ATCC29522 was suppressed completely at the first day of storage in sample likeYN24-N17, with pH of 4.36, and inhibited from 105 to 102 CFU/mL in sampleYN24. The decrease of the initial count of E.coli ATCC25922 from 105-102CFU/mL illustrates the antagnostic effect of the probiotic cultures on the pathogenic organisms which was due to pH and higher viable count of probiotics.

However, E.coli ATCC25922 completely disappeared at the second day of storage with  increased probiotic counts of 108 CFU/mL, when pH of probiotic yoghurt samples were not significantly different from each other at P≤0.05.  A similar study was reported by Kasimoglu and Akgun (2004), indicating that there was total suppression of E. coli within 48 hr after storage of the milk inoculated with 106 CFU/mL of probiotics. This could be due to the low pH below 4.39. The variable results of most authors could be strain dependent. Moreover, acid survival of food pathogens and their acid adaptation can enhance the survival of these organisms in acidic foods like yoghurt during fermentation.


 

 

Table 4: Antagonistic effect of probiotic yoghurt against Escherichia coliATCC25922(105 CFU/mL)  under cold storage (4oC)

 

Storage time(days)                             1

Bacterial count(CFU/mL)

 

 

              2

 

 

 

Samples

 

 

 

 

 

Codes

pH

ECC

pH

ECC

PCC

YN24

4.40a

1.4x103a

4.37a

-

2.4x107b

yn24 *

4.40a

-

4.36a

-

2.9x107b

YN17

4.41a

2.4x103a

4.37a

-

1.4 x107b

yn17

4.40a

-

4.37a

-

2.9x107b

YN10

4.39ab

1.8x102b

4.37a

-

3.2x107b

yn10

4.39ab

-

4.36a

-

3.8x107b

YN1

4.40ab

1.7x103a

4.39a

-

3.6x107b

yn1

4.38ab

-

4.38a

-

4.4x107b

YN24-N17

4.37b

-

4.36a

-

2.1x108a

yn24-n17

4.36b

-

4.36a

-

2.9x108a

YN24-N10

4.36b

-

4.35a

-

1.8x108a

yn24-n10

4.36b

-

4.36a

-

2.6x108a

YN24-N1

4.37b

-

4.36a

-

1.5x108a

yn24-n1

4.36b

-

4.35a

-

2.7x108a

YN17-N10

4.37b

-

4.35a

-

2.4x108a

yn17-n10

4.35b

-

4.35a

-

2.9x108a

YN17-N1

4.36b

-

4.35a

-

1.6x108a

yn17-n1

4.35b

-

4.34a

-

1.7x108a

YN10-N1

4.36b

-

4.35a

-

3.2x107a

yn10-n1

4.35b

-

4.35a

-

1.5x108a

Means with the same alphabets within a column are not significantly different at P≤0.05 using Duncan Multiple Range Test (DMRT). Data collected were represented as “Means of duplicates. Standard Deviation (SD)”not shown.

- = Not viable, initial and inoculum size of E. coliATCC25922= 105 CFU/mL,

ECC =E coli count, PCC=Probiotic cultures count, inoculum size of starters=106 CFU/mL,

*All small letters samples (yoghurt without pathogen)

*Samples with capital letters (yoghurt inoculated with E coliATCC25922).

 

 

 


CONCLUSIONS

 

The probiotic starters in the yoghurt were able to inhibit the growth, and suppressed selected food borne pathogens within a short period of 24 hr, suggesting that better ones could be useful to prevent or treat illness caused by pathogenic organisms, and also as preservatives in food and pharmaceutical industries.

 

 

ACKNOWLEDGEMENTS

 

I would like to express my gratitude to Professor A. A. Onilude, Department of Microbiology, University of Ibadan, Ibadan for his valuable supervision, and Dr.S.M. Wakil for her technical assistance and guidance.

 

 

REFERENCES

 

Al-Delaimy, K. S. and Hamamdeh, Y. M. (2013).   Inhibition of  Staphylococcus aureus by lactic acid bacteria and /or Bifidobacterium lactis during milk fermentation and storage. J. Microbio. Biotechnol. Food Sci. 2(4): 2406-2419.      

APHA, (2004). Standards Methods for the examination of dairy products.17thEdn. American Public Health Association Inc, Washington, DC,USA.

Bachrouri, M., Quinto, E. J. and  Mora, M.T. (2006). Survival of Escherichia coli O157:H7 during storage of yoghurt at different temperatures. J. Food Sci. 67 (5):1899-1903

Bibbal, D.,  Kerouredan, M.,  Loukiadis, E., Scheutz, F., Oswald, E. and Brugere, H. (2014). Slaughterhouse effluent discharges into rivers not responsible for environmental occurrence of enteroaggregative Escherichia coli. Veter. Microbio.168: 451-45.

Brant, R. J. and Todd, R. K. (2014). Impact of genomics on the field of probiotic research: historical perspectives to modern paradigms. Tot. Env. 106:141-156.

CDC, (2016)." Diarrhea: Common Illness, Centers for Disease Control and Prevention. Atlanta, USA, Pp.4

Donkor, O. N., Henriksson, A., Vasiljevic, T. and Shah, N. P. (2006). Effect of acidification on the activity of probiotics in yoghurt during cold storage. Internet. Dairy. J. 16: 1181-1189.

Evariste, B., Assèta, K., KuanAbdoulaye, T., Touwendsida, S. B., Hadiza, B. I., Soutongnooma, C. B., Isidore, J., Ouindgueta, B., Saidou, K., Cheikna, Z., Alfred, S. T. and Nicolas, B. (2017). Characterization of Diarrheagenic Escherichiacoli Isolated in Organic Waste Products (Cattle Fecal Matter, Manure and, Slurry)from Cattle’s Markets in Ouagadougou, Burkina Faso. Int. J. Environ. Res. Public Health.14: 1100

Gopalakrishnan,V. (2018).The influence of the gut microbiome on cancer, immunity and cancer immunotherapy, and Cancer cell. Sci. Am. 33(4) 570-580

IS0. (2003).Yoghurt: Enumeration of characteristics microorganisms by colony count technique at 37 0C. IstEdn., International Standard Organisation, Brussels, Belgium.

ISO. (2004). Horizontal Method for Detection and Enumeration of L. monocytogenes. 2ndEdn, International Standard Organisation, Brussels, Belgium.

Kasimoglu, A. And Akgun, S. (2004). Survival of E. coli O157:H7 in the processing and post-processing stages of acidophilus yoghurt. Internet.  J. Food Sci.Technol. 39: 563-568.

Mohammed, S., Ahlgren, J. A. and Horne, D. (2016). Structural characterization and biological activities of an exopolysaccharide kefir produced by Lactobacillus kefiranofaciens WT-2B(T). J. Agric. Food Chem. 52: 5533-5538.

Nassib, T. A., El- din, M. Z. and  El- Sharoud, W. M. (2006). Effect of thermophilic lactic acid bacteria on the viability of Salmonella serovartyphimurium PT8 during milk fermentation and preparation of buffalo’s yoghurt. Internet. Dairy. Technol.  59:29-34.

Nikolic, M., Terzic-Vidojevic, A., Jovcic, B., Begovic, Golic, N. and Topisirovic, L. (2008). Characterization of lactic acid bacteria isolated from Bukuljac, a homemade goat’s milk cheese. Internet. J. Food Microbio.122:162-170.

Rahmann, M., Gul, S. and Farooqi, W. (1999). Selection of starter culture for yoghurt preparation and its Antibacterial of Activity. Pak. J. Bio sci. 2: 131-133.

Rebello, R. C. and Regua-Mangia, A. H. (2014). Potential enterovirulence and antimicrobial resistance in Escherichia coli isolates from aquatic environments in Rio de Janeiro, Brazilian Science. Tot. Env. 490 :19–27

Ting, T. L. and X, Z. (2019). Lactobacillus spp as probiotics for prevention and treatments of enteritis in the lined seashore. Sci. Am. 503:16-25

Tribe, I. G., Cowell, N. D., Cameron, Peter.and Cameron, S. (2014). "An outbreak of Salmonella typhimurium phage type 135 infection linked to the consumption of raw shell eggs in an aged care facility". Communi. Dis. Intelli. 26 (1): 38–90.

Tsegaye, M. and Ashenafi, M. (2005). Fate of Escherichia coli O157:H7 during the processing and storage of Ergo and Ayib, traditional Ethiopian dairy products. Internet. J. Food Microbio. 103: 325-334

Wang ,Y. K.,  Li., S. N, Lu, C. S,  Peing, D. S.,  Su, Y. C, Wu, D. C., Jan, C. M., Lai, C. H., Wang, T. N. and Wang, W. M. (2004). Effect of injecting Lactobacillus and Bifidobacterium containing yoghurt in subjects with colonized  Helicobacter pylori. Am. J. Clin. Nutr. 80(3): 737-4.


 

 

Cite this Article: Aforijiku, S; Onilude, AA; Wakil, SM (2019). Antagonistic effect of developed probiotic yoghurt against some selected food-borne pathogens during cold storage. Greener Journal of Biological Sciences, 9(2): 22-28, http://doi.org/10.15580/GJBS.2019.2.071719137.