Abstract
This study investigated growth characteristics and antibacterial properties of probiotics in fermented coconut milk (CM) against Streptococcus pyogenes. A total of eight probiotics were screened for their survivability in CM. Antibacterial test against S. pyogenes was carried out on strain with highest growth rate. The survivability of probiotics in CM is strain dependent with S. salivarius ATCC 13419 showing the highest growth rate. Titratable acidity of the CM increased significantly while pH decreased significantly upon 9 h fermentation. The antibacterial properties of CM fermented with S. salivarius ATCC 13419 and K12 against S. pyogenes enhanced by 60.60% and 67.69%, respectively, compared to non-fermented CM. Their ability to metabolise carbohydrates and fats in CM was proven where alpha-glucosidase activity of S. salivarius ATCC 13419 and K12 was 22.42 ± 1.73 and 24.92 ± 7.22 unit/L, respectively, whereas, lipase activity was 1498.29 ± 48.50 and 1749.90 ± 254.28 unit/L, respectively. Lipolytic activity of these strains was further evidenced by GC–MS results whereby lauric acid content (potent antibacterial substance) in CM fermented with S. salivarius ATCC 13419 and K12 increased significantly by 5.03% and 10.74%, respectively. In conclusion, fermented CM provided a new alternative of non-dairy functional product with antibacterial potential against S. pyogenes.
Keywords: Probiotic, Coconut milk, Fermentation, Antibacterial properties
Introduction
Pharyngitis, also known as sore throat is one of the most common diseases which may be triggered by either bacterium like Streptococcus pyogenes or viruses, typically rhinovirus. Such disorder could lead to acute infection of upper respiratory tract, affecting the throat and respiratory mucosa (Kenealy 2015). In most of the bacterial-induced cases, antibiotics, for instance, penicillin is prescribed to patient to subside and reduce the symptoms of bacterial-induced pharyngitis. However, antibiotics may lead to certain side effects like nausea, vomiting, diarrhoea and moreover, widespread and misuse of such antibiotics causes a more severe extent, bacterial resistance (Kenealy 2015). Thus, studies are constantly carried out to look into solutions to overcome this issue. It is now understood that the beneficial effects of probiotics are not just limited to gastrointestinal tract but also on other human sites like respiratory tract, oropharyngeal tract and urogenital tract (Popova et al. 2012). Recent studies have suggested that the potential of probiotics in antagonising S. pyogenes, considering their ability to restore lost bacteria, maintenance of immune homeostasis and inhibitory effect on the growth of pathogens (Popova et al. 2012). The use of probiotics in prevention against S. pyogenes infection thus showed a promising effect. However, to date, there is still very limited literature available demonstrating the antibacterial effect of probiotics against S. pyogenes.
Probiotics are live microorganisms that confer health benefits on the host when an adequate amount is consumed (FAO/WHO 2001). Essentially, these microorganisms in the food product need to be alive, active as well as achieve a minimum amount of 107 CFU/mL or gram to be deemed effective and thus able to exert a health effect on the host. The general probiotic species that are commonly incorporated into food matrix are those from the family of Lactobacillus and Bifidobacterium as they are generally regarded as safe (GRAS) but certain strains from the Streptococcus genus also showed their potential as a probiotic functional food. For example, Streptococcus salivarius, an oral probiotic which is well established and remains as predominant commensal inhabitant in the human oral cavity has an important role in maintaining the balance and health of the oral ecology (Kaci et al. 2013). A study by Tagg and Dierksen (2003) showed that the S. salivarius K12 isolated from a child is able to prevent colonisation of S. pyogenes. As such, we have included certain strains of oral probiotic in the present study to study their antibacterial potential against S. pyogenes.
In the past, probiotics were generally incorporated into dairy milk to be delivered to human. Lately, there has been a rising trend for non-dairy probiotic food due to some major drawbacks from dairy-based products for example consumers with lactose intolerance and those who practice vegan diet (Lee et al. 2013). Furthermore, there are also concerns on the cholesterol content of dairy milk in which consumers ought to seek for alternative to increase the variety of probiotic functional foods. Hence, the use of non-dairy ingredients such as soymilk and coconut milk as a replacement for incorporation of probiotics has been explored and studied in order to cope with consumers’ demand (Aboulfazli et al. 2014).
Coconut milk, an indispensable ingredient in the South East Asia delicacy, refers to the aqueous substance extracted from solid coconut endosperm and is essentially free from fibre (Seow and Gwee 1997). An earlier study has demonstrated that the consumption of coconut milk may aid in the treatment of obesity, insulin resistance, type 2 diabetes, hypertension, Alzheimer’s Disease (Fernando et al. 2015). Recently, coconut milk has been considered as a substitute for dairy milk for probiotic incorporation because it is highly nutritious with abundance of fats, protein, carbohydrates, minerals and vitamins that are able to promote probiotics growth (Yuliana et al. 2010). Noticeably, lauric acid, the predominant fatty acid present in coconut milk possesses antibacterial properties against pathogenic bacteria (Lakshmi et al. 2017; Pehowich et al. 2000). The antibacterial effect is magnified when the lauric acid is utilised by human body and converted into its derivative monolaurin which possesses more potent antibacterial properties (Batovska et al. 2009).
To date, the development of probiotic food using plant-based milk is relatively new and utilisation of coconut milk as fermentative medium by probiotic is still very limited (Jeske et al. 2018; Shana et al. 2015). It is thus believed that the rich nutrient contents of coconut milk could be a suitable matrix to support the probiotic growth. Therefore, this study aimed to evaluate the growth of probiotics in coconut milk and their relative antibacterial potential against S. pyogenes to produce a novel probiotic functional food.
Materials and methods
Probiotic strains and the pathogen used in this study were obtained from culture collection of Taylor’s University and are listed in Table 1. The Lactobacillus strains were propagated three times (10% inoculum) in de Man, Rogosa & Sharpe (MRS) broth (Merck, Darmstadt, Germany) and incubated anaerobically at 37 °C for 24 h, whereas, Streptococcus strains were sub-cultured for three times (10% inoculum) and incubated aerobically for 24 h in Brain Heart Infusion (BHI) broth (Himedia, Mumbai, India) at 37 °C. The stock cultures were kept frozen at − 80 °C in the presence of 40% glycerol.
Table 1.
List of probiotic strains and pathogen used in the study
| Bacteria species | Strains |
|---|---|
| Lactobacillus casei | ATCC 393 |
| Lactobacillus fermentum | ATCC 14931 |
| Lactobacillus fructivorans | 8 |
| Lactobacillus rhamnosus | ATCC 53103 |
| Lactobacillus acidophilus | ATCC 314 |
| Streptococcus salivarius | K12 |
| ATCC 13419 | |
| TUCC 1253 | |
| Streptococcus pyogenes (pathogen) | ATCC 19615 |
Preparation and fermentation of coconut milk
Freshly grated raw coconut milk was purchased from same local store (Kuala Lumpur, Malaysia). Coconut milk was filtered twice with cheese cloth and subjected to pasteurisation at 80 °C for 10 min prior to chilling to 4 °C (Aboulfazli et al. 2014). Coconut milk was prepared freshly each time and was used immediately. Fermentation of coconut milk was done by inoculation of 10% (v/v) activated probiotic culture (OD600 = 1) into coconut milk. The fermentation process was carried out for 9 h at 37 °C aerobically.
Viability of probiotic in fermented coconut milk
The viable cell count of each probiotic in coconut milk before and after fermentation was determined by pour plating method. The samples were diluted with 0.9% (w/v) saline (R&M Chemicals, London, UK) and plated on MRS agar for Lactobacillus strains and BHI agar for Streptococcus strains. The MRS plates were incubated anaerobically while BHI plates were incubated aerobically, both at 37 °C for 24 h.
Antibacterial property of probiotics against oral pathogen
Antibacterial property against S. pyogenes was done on two probiotics with the highest growth rate after 9 h of fermentation using disc diffusion method according to Lakshmi et al (2017). The S. pyogenes was grown on BHI agar in the presence of coconut milk impregnated sterilised blank disc (ThermoFisher, Massachusetts, USA). The antibacterial property was determined by measuring the diameter of clear zones around the disc. Concentration of the probiotics and S. pyogenes were standardised to OD600 of 1.0 A and 0.2 A, respectively, using BHI and MRS broth accordingly with UV/Vis spectrophotometer (ThermoFisher, Massachusetts, USA) at wavelength, λ = 600 nm. On a sterile BHI agar, 100 µL of S. pyogenes was dispensed and spread evenly across the plate with 20 µL fermented coconut milk on the sterilised disc. The plates were then incubated aerobically at 37 °C for 24 h. The presence of clear inhibition zones was determined and the diameter of zone of inhibition was measured to the nearest mm using a ruler. Antibiotic penicillin and non-fermented coconut milk were used as control sample.
Chemical analysis of fermented coconut milk
The pH of coconut milk was measured with a calibrated pH meter (Eutech pH, Singapore) before and after fermentation. Titratable acidity (TA) was determined by titrating 10 mL of fermented coconut milk with 0.1 N sodium hydroxide (R&M Chemicals, London, UK), using phenolphthalein as an indicator (Aboulfazli et al. 2014). The amount of TA was expressed as the percentage of lactic acid by multiplying the average titre with the conversion factor of lactic acid (0.09008).
Fatty acid profile of fermented coconut milk
The fatty acid profile of coconut milk before and after fermentation was determined using GC–MS (Agilent Technologies, California, USA) (AOAC 2012). The fermented coconut milk was first converted into fatty acid methyl esters (FAME) before injecting into GC column. Free fatty acid in coconut milk was extracted using modified Mojonnier method (AOAC 2006). The extracted coconut oil was methylated into FAME by mixing 1 g of oil with 0.435 mL of 95% HPLC graded methanol (Merck, Darmstadt, Germany) and 0.01 g of crushed sodium hydroxide as catalyst and subsequently filtered using 0.22 μm filter (Minisart, Sartorius, Germany). GC–MS analysis was performed using capillary column Hp-5 ms; 30 m × 0.25 mm ID, film 0.25 (Agilent Technologies, California, USA). The injector was held at 325 °C. The helium and nitrogen gas were used as carrier gas with a flow rate of 1 mL/min and a split ratio of 1:10. The injection volume was 1 μL. Fatty acids were identified by comparing their retention time with the in-built MS library. Results were expressed as relative percentage of each fatty acid.
Alpha-glucosidase activity of probiotic in fermented coconut milk
Alpha-glucosidase activity of probiotics was measured at 37 °C using alpha-glucosidase assay kit (Sigma Aldrich, St. Louis, Missouri, USA) based on colorimetric method. Roughly, the coconut milk was mixed with a master reaction which consists of assay buffer and p-nitrophenyl-α-D-glycopyranoside (α-NPG) as substrate. The initial absorbance was measured with microplate reader coupled with Gen5 Software (Biotek Instruments, Vermont, USA) at wavelength of 405 nm followed by incubation at 37 °C for 20 min. Then, the final absorbance was measured again. The formation of colorimetric product at 405 nm is proportional to the alpha-glucosidase activity present. The specific activity was expressed as units/L where one unit of alpha-glucosidase is the amount of enzyme that catalyses the hydrolysis of one μmole α-NPG substrate per minute at pH 7.0.
Lipase activity of probiotics in fermented coconut milk
Lipase activity was determined using Lipase Activity Assay Kit (Elabscience, Texas, USA) based on calorimetric method with triglycerides as substrate. The coconut milk was pre-treated at 37 °C for 30 min. The rate of hydrolysis for 10 min at 37 °C was measured in pH 7.0 tris buffer at wavelength of 420 nm in UV/Vis spectrophotometer (Perkin Elmer, Massachusetts, United States). The specific activity was expressed as unit/L where one unit of lipase activity is defined as one μmole of substrate that consumed by one L of serum in one minute at 37 °C.
Statistical analysis
The IBM SPSS 25 Inc. Software (Chicago, United States) was used to analyse the data collected from the test carried out. Data were compared using one-way analysis of variance (ANOVA) unless otherwise stated. Mean value comparisons were performed using Tukey test. The experiments were performed in triplicate of two separate runs (n = 2). Results of each analysis were expressed as mean ± standard deviation values with a statistical significance level of α = 0.05.
Results and discussion
Growth of probiotics in coconut milk before and after fermentation
While selecting the probiotic bacteria for fermentation, one of the important criteria is that they must be able to be produced in large scale and at the same time able to survive in the food products prior to consumption by the consumers (Araújo et al. 2012). To date, the knowledge on fermentation of coconut milk is rather limited, especially fermentation with probiotic bacteria (Lee et al. 2013). Among these, Yuliana et al (2010) and Mauro and Gracia (2019) have demonstrated that the rich nutrients in coconut milk which include sugars, fats and minerals are able to support and maintain the growth of selected probiotics such as L. acidophilus and L. reuteri while Lakshmi et al (2017) suggested that coconut milk could be used as a suitable alternative to cow’s milk to produce coconut milk kefir. However, other species of beneficial bacteria strains such as Streptococcus salivarius were rarely studied for survivability in coconut milk.
The viability and growth rate of all the eight probiotic strains in coconut milk were evaluated before and after fermentation (Table 2). In general, all eight strains of probiotic showed a significant increase in growth after the fermentation (p < 0.05), ranging from 0.23 to 0.75 log CFU/mL (p < 0.05). Among all tested probiotic strains, S. salivarius ATCC 13419 had shown the highest viability as well as growth rate. The increase of viable cells in probiotics indicated that the nutritious coconut milk could be an ideal fermentative source to support the growth of the probiotics (Szparaga et al. 2019). However, past studies suggested that the ability to grow in medium is strain dependent, hence, the results showed a difference in viability and growth rate in each of the probiotic strains.
Table 2.
Viability and growth rate of probiotic (log CFU/mL) in fermented coconut milk
| Probiotics | Mean viable count (log CFU/mL) | Growth rate | |
|---|---|---|---|
| Before | After | ||
| S. salivarius ATCC 13419 | 12.91 ± 0.06a | 13.66 ± 0.05A | 0.75 ± 0.01a |
| S. salivarius K12 | 13.12 ± 0.01a | 13.42 ± 0.02A | 0.30 ± 0.01b |
| L. casei ATCC 393 | 8.75 ± 0.19b | 9.45 ± 0.10B | 0.70 ± 0.12a |
| S. salivarius TUCC 1253 | 8.66 ± 0.45b | 8.89 ± 0.56B | 0.23 ± 0.11b |
| L. rhamnosus ATCC 53103 | 8.03 ± 0.14b | 8.76 ± 0.13B | 0.73 ± 0.01a |
| L. acidophilus ATCC 314 | 7.66 ± 0.22c | 8.32 ± 0.09C | 0.66 ± 0.13a |
| L. fermentum ATCC 14931 | 6.81 ± 0.08d | 7.21 ± 0.05D | 0.40 ± 0.13b |
| L. fructivorans 8 | 7.71 ± 0.20c | 8.15 ± 0.08C | 0.44 ± 0.01b |
Concentration of probiotics were adjusted to OD600 = 1.0 A by using BHI broth for Streptococcus strains and MRS broth for Lactobacillus
Before = before fermentation; After = after 9 h fermentation; Growth rate = Difference between the viability of probiotic in coconut milk before and after 9 h fermentation
Results are expressed as mean ± standard deviation of means; values are mean of triplicate from two different runs (n = 2) via paired one-way ANOVA test
Mean viable cell counts: Difference in letters in the same row indicated a significance difference (p < 0.05); Growth rate: Different letters in the same column indicated a significance difference (p < 0.05)
While the antibacterial properties of probiotics on gastrointestinal pathogens have been widely reported, their potential against oropharyngeal site is still scarce. Therefore, it is an interesting area to explore and widen the application of probiotic food. The two strains with the highest growth rate in the present study were S. salivarius ATCC 13419 and L. casei ATCC 393. Nevertheless, as past evidence suggested that S. salivarius K12 has positive antagonism effect on S. pyogenes, the bacteria known as the common cause of acute pharyngitis, hence, the strain K12 was included as a positive control in this study for further evaluation on antibacterial potential against S. pyogenes in coconut milk.
Chemical analysis of coconut milk before and after fermentation
The pH and TA of non-fermented and fermented coconut milk are shown in Table 3. Coconut milk before fermentation process has an initial pH level of 6.35 and TA of 0.19%. Upon fermentation at 37 °C, the pH of fermented coconut milk significantly decreased to a range from pH 3.96 to 4.98, whereas, the TA of fermented coconut milk significantly increase, ranging from 0.29 to 0.77%, respectively (p < 0.05).
Table 3.
The pH and titratable acidity (%) of fermented coconut milk before and after 9 h fermentation
| pH | Titratable acidity (%) | |
|---|---|---|
| Non-fermented coconut milk | 6.35 ± 0.02a | 0.19 ± 0.00 a |
| Fermented coconut milk | ||
| L. casei ATCC 393 | 4.62 ± 0.19b | 0.35 ± 0.002c |
| L. fermentum ATCC 14931 | 4.98 ± 0.27b | 0.73 ± 0.009f |
| L. rhamnosus ATCC 53103 | 4.46 ± 0.06c | 0.46 ± 0.010d |
| L. acidophilus ATCC 314 | 3.96 ± 0.13d | 0.77 ± 0.019e |
| L. fructivorans 8 | 4.49 ± 0.09c | 0.60 ± 0.016g |
| S. salivarius TUCC 1253 | 4.55 ± 0.17c | 0.31 ± 0.005b |
| S. salivarius ATCC 13419 | 4.51 ± 0.09c | 0.29 ± 0.023b |
| S. salivarius K12 | 4.71 ± 0.09b | 0.38 ± 0.010c |
Results are expressed as mean ± standard deviation of means; values are mean of triplicates from two different runs (n = 2) via one-way ANOVA test
Difference in letters in the same column represent a significance difference (p < 0.05)
It is understood that the main sugars presented in coconut milk are sucrose and some starch which will be utilised as nutrient source for probiotic growth during fermentation (Yuliana et al. 2010). The significant decrease in pH accompanied with elevated TA in fermented coconut milk indicated that all the probiotics selected actively multiply by utilising the rich carbohydrates in coconut milk and subsequently produce organic acids such as lactic acid and acetic acids as by-products upon fermentation (Szparaga et al. 2019). The accumulation of these organic acids that leads to decrease in pH and increase in TA proportionally had demonstrated the capability of the probiotics to adapt and survive in coconut milk environment.
Antibacterial properties of fermented coconut milk against S. pyogenes
Antibacterial test was performed with the aim to re-establish a balance ratio in oral community by replacing the pathogens in order to maintain a good oropharyngeal health. Two strains, S. salivarius 13419 and S. salivarius K12 were further tested for their antagonistic effect against S. pyogenes. The antibacterial activity results of non-fermented and fermented coconut milk against S. pyogenes are presented in Table 4. Non-fermented coconut milk without addition of probiotic also displayed an antibacterial effect towards S. pyogenes (7.19 ± 0.24 mm). Upon fermentation, coconut milk fermented with the S. salivarius strains showed a significant increase in antibacterial activity against S. pyogenes by 60.60 to 67.69% compared to non-fermented coconut milk (p < 0.05), ranging from 18.25 to 22.25 mm.
Table 4.
Mean diameter of inhibition zones (mm) of non-fermented coconut milk, coconut milk fermented with S. salivarius ATCC 13419 and S. salivarius K12 and penicillin against S. pyogenes
| Samples | Mean diameter of inhibition zones (mm) |
|---|---|
| Non-fermented coconut milk | 7.19 ± 0.24a |
| Penicillin | 13.00 ± 0.00c |
| S. salivarius K12 | 22.25 ± 4.99b |
| S. salivarius ATCC 13419 | 18.25 ± 1.71b |
Antibiotic concentration tested: 2.5 μg penicillin
Results are expressed as mean ± standard deviation of means; values are mean of triplicate from two different runs (n = 2) via one-way ANOVA test
Difference in letters in the same column represents a significance difference (p < 0.05)
The antibacterial activity of fermented coconut milk could be associated with the production of organic acids which resulted from metabolism of carbohydrates by the S. salivarius (Lakshmi et al. 2017). This is supported by our results which showed an elevated TA level in fermented coconut milk. These organic acids enter into bacterial cytoplasm, consequently lower the intracellular pH of the pathogen and eventually lead to cell death (Bajaj et al. 2015). Furthermore, it was observed that coconut milk fermented with S. salivarius K12 exhibited a greater antibacterial effect (22.25 ± 2.50 mm) than S. salivarius ATCC 13419-fermented coconut milk (18.25 ± 1.71 mm) which to an extent significantly greater than 2.5 μg penicillin antibiotic (13.00 ± 0.00 mm) (p < 0.05). This could be due to its antagonistic mechanisms against pathogens which is the ability to secrete antimicrobial substances such as salivaricin A2 and B by S. salivarius K12 (Bajaj et al. 2015; Hyink et al. 2007).
Our results indicated that coconut milk itself possesses antibacterial properties against S. pyogenes but is much weaker than the fermented counterparts. The presence of lauric acid in coconut milk contributes to the antibacterial properties (Khoramnia et al. 2013). In fact, coconut milk is rich in fats composed of mainly medium chain fatty acids (MCFAs) and the most abundant MCFA is lauric acid, making up about 50% of the total fatty acid content in coconut milk (Khoramnia et al. 2013). During fermentation, the probiotic is able to convert the lauric acids into its bioactive derivatives which possess antibacterial ability to protect the host from certain viral and bacterial infections (Pehowich et al. 2000). Hence, this could be the reason for the significant enhance of antibacterial activities of fermented coconut milk compared to the non-fermented control in our study. In regards to this, we further study the fatty acid profile of fermented coconut milk via GC–MS. The ability of the oral probiotic S. salivarius to counteract with S. pyogenes shown in this study renders them to be a potential approach in eliminating S. pyogenes, which could then be used to maintain a healthy oropharyngeal tract.
Fatty acid profile of fermented coconut milk
Coconut milk is an important source of MCFA which shown to exhibit antimicrobial properties (Khoramnia et al. 2013). Guerzoni et al (2001) reported certain strains of lactobacilli are able to grow in the presence of MCFAs possibly due to their lipolytic activities. Several studies also reported that lactic acid bacteria (LAB) may contribute to the production of free fatty acids through lipolysis of milk fat (Kim and Liu 2002; Yadav et al. 2007). Although such activity in LAB has been reported, they were generally deemed to possess weak lipolytic activity (Esteban-Torres et al. 2015). In addition, Guan et al (2020) also demonstrated that there were only limited strains of probiotics with lipolytic activity. The current study showed that the selected probiotic strains were able to grow well in coconut milk which contains rich amount of milk fats. This could be due to the enzyme lipase in probiotics that hydrolyses coconut milk fat and releases the fatty acids.
With this, we further analysed the MCFAs profile of non-fermented and fermented coconut milk (Table 5). There was a significant decrease in caprylic acid and capric acid levels after fermentation (p < 0.05). However, the result was in contrast with past study by Parfene et al (2013) who reported an increased in caprylic acid and capric acid after 3 days of fermentation. On the other hand, the lauric acid content in fermented coconut milk with S. salivarius 13419 and K12 strain had increased significantly (p < 0.05) by 5.03% and 10.74%, respectively after fermentation. The finding is in good agreement with previous study which also demonstrated a significant increase in lauric acid content after fermentation through enzymatic hydrolysis of the substrates (Khoramnia et al. 2013; Parfene et al. 2013).
Table 5.
Fatty Acid Methyl Ester (FAME) composition of non-fermented and fermented coconut milk
| Fatty Acid Methyl Ester (FAME) | FAME Composition (%) | ||
|---|---|---|---|
| Non- fermented Coconut Milk | S. salivarius K12 | S. salivarius ATCC 13419 | |
| Caprylic acid, methyl ester (C8:0) | 22.23 ± 0.77a | 6.32 ± 0.06b | 18.80 ± 0.33c |
| Capric acid, methyl ester (C10:0) | 10.14 ± 0.69a | 6.07 ± 0.07b | 9.10 ± 0.31c |
| Lauric acid, methyl ester (C12:0) | 55.46 ± 0.33a | 62.14 ± 0.32b | 58.40 ± 0.09c |
Results are expressed as mean ± standard deviation of means; values are mean of triplicates from two different runs (n = 2) via one way-ANOVA test
Different letters in the same row indicated significance difference (p < 0.05)
The increase in lauric acid is indeed desirable as past study has demonstrated that lauric acid possesses the strongest antibacterial effect among all the fatty acids (Khoramnia et al. 2013). Taking into account of the antibacterial effect of the lauric acid in coconut milk, the increase in lauric acids level after fermentation may account for the increased antibacterial effect of the fermented coconut milk. Hence, we postulated that the enhanced antibacterial activity against S. pyogenes could be due to the synergistic effect of coconut milk and the Streptococcus strains.
Enzymatic activities of probiotics in fermented coconut milk
The results of alpha-glucosidase and lipase activities of S. salivarius ATCC 13419 and K12 strain are presented in Table 6. Based on the result, the alpha-glucosidase and lipase activities for both Streptococcus strains had increased significantly after fermentation (p < 0.05), clearly indicating an active metabolic process in the probiotic cells throughout fermentation of coconut milk. Particularly, the alpha-glucosidase activity of S. salivarius ATCC 13419 and K12 was 22.42 ± 1.73 unit/L and 24.92 ± 7.22 unit/L, respectively, whereas, the lipase activity was 1498.20 ± 48.05 unit/L and 1747.90 ± 254.28 unit/L, respectively.
Table 6.
Alpha-glucosidase and lipase activities of probiotics in coconut milk after 9 h of fermentation
| Enzyme | Enzymatic activities (unit/L) | |
|---|---|---|
| S. salivarius K12 | S. salivarius ATCC 13419 | |
| Alpha-glucosidase | 24.92 ± 7.22a | 22.42 ± 1.73b |
| Lipase | 1747.90 ± 254.28a | 1498.20 ± 48.05b |
Results are expressed as mean ± standard deviation of means; values are mean of triplicates from two different runs (n = 2) via one way-ANOVA test
Difference in letters in the same row represents a significance difference (p < 0.05)
During fermentation, probiotics may secrete hydrolytic enzymes such as alpha-glucosidase and lipase to break down the carbohydrate sources and fat contents in coconut milk for growth. For instance, alpha-glucosidase is responsible to hydrolyses the sucrose and starches in coconut milk, subsequently produces lactic acid as the end product which causes a reduction in the pH level (Choo et al. 2020). While on the other hand, lipase released by probiotics catalyses the lipolysis of coconut milk fat, thereby releasing free fatty acids (Parfene et al. 2013). Our results also showed that S. salivarius K12 has a significant higher alpha-glucosidase and lipase activities as compared to S. salivarius ATCC 13419 (p < 0.05), indicating a higher metabolic rate in coconut milk fermented with strain K12. These were further supported by the significant higher titratable acidity and lauric acid content in coconut milk fermented with K12 strain. Nevertheless, it is important to note that the enzymatic activities of probiotics are strain dependent owing to the difference in their ability in utilising the nutrient sources.
Conclusion
In conclusion, the significant increase in growth rate of S. salivarius ATCC 13419 and K12 in coconut milk after 9 h of fermentation proved that coconut milk could serve as an ideal fermentative medium for the incorporation of probiotics owing to the rich nutrient profiles in coconut milk which would able to sustain the growth of probiotics. Results also showed that both of the Streptococcus strains are able to release enzyme alpha-glucosidase and lipase to break down the carbohydrate sources and fats in coconut milk. Moreover, the antibacterial effect of both Streptococcus-fermented coconut milk against S. pyogenes was significantly enhanced after fermentation due to the synergic effect of fermentation of probiotics and the presence of lauric acid in coconut milk which possesses antibacterial properties. Further GC–MS analysis on MCFAs profile on fermented coconut milk confirmed that the lauric acid content increased significantly after fermentation. This is partly due to their lipolytic activity that hydrolyses the coconut milk fat, subsequently releases free lauric acid and contributes to the enhancement of antibacterial property against S. pyogenes. Hence, both coconut milk fermented with S. salivarius K12 and ATCC 13419 showed a promising antibacterial effect against S. pyogenes which can be considered as an ideal non-dairy probiotic functional food for consumers to maintain a healthy oropharyngeal tract.
Acknowledgements
This research was carried out with the infrastructure and facilities support of Taylor’s University Malaysia. We acknowledge the financial support of Taylor’s University Research Grant (TRGS/MFS/2018/SBS/001).
Author’s contribution
CEH carried out the work and wrote the MS; SKY supervised the work, corrected and edited the manuscript; JAE and CSK conceived and provided idea and research framework.
Funding
This research was supported by Taylor’s University Research Grant (TRGS/MFS/2018/SBS/001).
Availability of data and material
All data generated or analysed during this study are included in this published article.
Declarations
Conflicts of interest
The authors declare that they have no conflict of interest.
Footnotes
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Contributor Information
Ching Enn Han, Email: chingenn@gmail.com.
Joo-Ann Ewe, Email: jooannewe@nami.org.hk.
Chee-Sian Kuan, Email: cs_sam85@yahoo.com.my.
Siok Koon Yeo, Email: SiokKoon.Yeo@taylors.edu.my.
References
- Aboulfazli F, Baba A, Misran M. Effect of vegetable milks on the physical and rheological properties of ice cream. Food Sci Technol Res. 2014;20:987–996. doi: 10.3136/fstr.20.987. [DOI] [Google Scholar]
- AOAC (2006) Official methods of analysis. 18th edn, Association of Official Analytical Chemists, Gaithersburg, MD
- AOAC (2012) Official methods of analysis. 19th edn, Association of Official Analytical Chemists, Gaithersburg, MD
- Araújo E, dos Santos Pires A, Pinto M, Jan G, Carvalho A. Probiotics in dairy fermented products. In: Rigobelo E, editor. Probiotics. Croatia: InTech; 2012. [Google Scholar]
- Bajaj B, Claes I, Lebeer S. Functional mechanisms of probiotics. J Microbiol Biotechnol Food Sci. 2015;4:321–327. doi: 10.15414/jmbfs.2015.4.4.321-327. [DOI] [Google Scholar]
- Batovska D, Todorova I, Tsvetkova I, Najdenski H. Antibacterial study of the medium chain fatty acids and their 1-monoglycerides: Individual effects and synergistic relationships. Pol J Microbiol. 2009;58:43–47. [PubMed] [Google Scholar]
- Choo S, Yap K, Yap W, Ewe J. Soy fermentation by orally isolated putative probiotic Streptococcus salivarius for healthy oral. J Int Oral Health. 2020;12:33–40. doi: 10.4103/jioh.jioh_196_19. [DOI] [Google Scholar]
- Esteban-Torres M, Mancheño J, de las Rivas B, Muñoz R. Characterization of a halotolerant lipase from the lactic acid bacteria Lactobacillus plantarum useful in food fermentations. LWT. 2015;60:246–252. doi: 10.1016/j.lwt.2014.05.0633. [DOI] [Google Scholar]
- Fernando W, Martins I, Goozee K, Brennan C, Jayasena V, Martins R. The role of dietary coconut for the prevention and treatment of Alzheimer's disease: potential mechanisms of action. Br J Nutr. 2015;114:1–14. doi: 10.1017/s0007114515001452. [DOI] [PubMed] [Google Scholar]
- Food and Agriculture Organization of the United Nations, World Health Organization (FAO/WHO) (2001) Evaluation of health and nutritional properties of probiotics in food including powder milk with live lactic acid bacteria. Córdoba, Argentina
- Guan C, Tao Z, Wang L, Zhao R, Chen X, Huang X, Su J, Lu Z, Chen X, Gu R. Isolation of novel Lactobacillus with lipolytic activity from the vinasse and their preliminary potential using as probiotics. AMB Express. 2020;10:1–11. doi: 10.1186/s13568-020-01026-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Guerzoni M, Lanciotti R, Vannini L, Galgano F, Favati F, Gardini F, Suzzi G. Variability of the lipolytic activity in Yarrowia lipolytica and its dependence on environmental conditions. Int J Food Microbiol. 2001;69:79–89. doi: 10.1016/s0168-1605(01)00575-x. [DOI] [PubMed] [Google Scholar]
- Hyink O, Wescombe P, Upton M, Ragland N, Burton J, Tagg J. Salivaricin A2 and the novel lantibiotic Salivaricin B are encoded at adjacent loci on a 190-kilobase transmissible megaplasmid in the oral probiotic strain Streptococcus salivarius K12. Appl Environ Microbiol. 2007;73:1107–1113. doi: 10.1128/aem.02265-06. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Jeske S, Zannini E, Arendt E. Past, present and future: the strength of plant-based dairy substitutes based on gluten-free raw materials. Food Res Int. 2018;110:42–51. doi: 10.1016/j.foodres.2017.03.045. [DOI] [PubMed] [Google Scholar]
- Kaci G, Goudercourt D, Dennin V, Pot B, Doré J, Ehrlich S, Renault P, Blottiere HM, Daniel C, Delome C. Anti-inflammatory properties of Streptococcus salivarius, a commensal bacterium of the oral cavity and digestive tract. Appl Environ Microbiol. 2013;80:928–934. doi: 10.1128/aem.03133-13. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kenealy T. Sore throat. Am Fam Physician. 2015;91:689–690. [PubMed] [Google Scholar]
- Khoramnia A, Ebrahimpour A, Ghanbari R, Ajdari Z, Lai O. Improvement of medium chain fatty acid content and antimicrobial activity of coconut oil via solid-state fermentation using a Malaysian Geotrichum candidum. BioMed Res Int. 2013 doi: 10.1155/2013/954542. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kim Y, Liu R. Increase of conjugated linoleic acid content in milk by fermentation with lactic acid bacteria. J Food Sci. 2002;67:1731–1737. doi: 10.1111/j.1365-2621.2002.tb08714.x. [DOI] [Google Scholar]
- Lakshmi T, Mary Pramela A, Iyer P. Anti-microbial, anti-fungal and anti-carcinogenic properties of coconut milk kefir. Int J Home Sci. 2017;3:365–369. [Google Scholar]
- Lee P, Boo C, Liu S. Fermentation of coconut water by probiotic strains Lactobacillus acidophilus L10 and Lactobacillus casei L26. Ann Microbiol. 2013;63:1441–1450. doi: 10.1007/s13213-013-0607-z. [DOI] [Google Scholar]
- Mauro C, Garcia S. Coconut milk beverage fermented by Lactobacillus reuteri: optimization process and stability during refrigerated storage. J Food Sci Technol. 2019;56:854–864. doi: 10.1007/s13197-018-3545-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Parfene G, Horincar V, Tyagi A, Malik A, Bahrim G. Production of medium chain saturated fatty acids with enhanced antimicrobial activity from crude coconut fat by solid state cultivation of Yarrowia lipolytica. Food Chem. 2013;136:1345–1349. doi: 10.1016/j.foodchem.2012.09.057. [DOI] [PubMed] [Google Scholar]
- Pehowich D, Gomes A, Barnes J. Fatty acid composition and possible health effects of coconut constituents. West Indian Med J. 2000;49:128–133. [PubMed] [Google Scholar]
- Popova M, Molimard P, Courau S, Crociani J, Dufour C, Le Vacon F, Carton T. Beneficial effects of probiotics in upper respiratory tract infections and their mechanical actions to antagonize pathogens. J Appl Microbiol. 2012;113:1305–1318. doi: 10.1111/j.1365-2672.2012.05394.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Seow C, Gwee C. Coconut milk: chemistry and technology. Int J Food Sci Tech. 1997;32:189–201. doi: 10.1046/j.1365-2621.1997.00400.x. [DOI] [Google Scholar]
- Shana SR, Roopa BS, Vijayendra SVN. Optimization of a novel coconut milk supplemented dahi – a fermented milk product of Indian subcontinent. J Food Sci Technol. 2015;52(11):7486–7492. doi: 10.1007/s13197-015-1825-0. [DOI] [Google Scholar]
- Szparaga A, Tabor S, Kocira S, Czerwińska E, Kuboń M, Płóciennik B, Findura P. Survivability of probiotic bacteria in model systems of non-fermented and fermented coconut and hemp milks. Sustainability. 2019;11:6093. doi: 10.3390/su11216093. [DOI] [Google Scholar]
- Tagg J, Dierksen K. Bacterial replacement therapy: adapting ‘germ warfare’ to infection prevention. Trends Biotechnol. 2003;21:217–223. doi: 10.1016/s0167-7799(03)00085-4. [DOI] [PubMed] [Google Scholar]
- Yadav H, Jain S, Sinha P. Production of free fatty acids and conjugated linoleic acid in probiotic dahi containing Lactobacillus acidophilus and Lactobacillus casei during fermentation and storage. Int Dairy J. 2007;17:1006–1010. doi: 10.1016/j.idairyj.2006.12.003. [DOI] [Google Scholar]
- Yuliana N, RanggaRakhmiati A. Manufacture of fermented coco milk-drink containing lactic acid bacteria culture. Afr J Food Sci. 2010;4:558–562. [Google Scholar]
Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Data Availability Statement
All data generated or analysed during this study are included in this published article.