Evaluation of antioxidant, antimicrobial and potential anticancer activities of papaya-based kombucha beverages on colon cancer

Traditional kombucha is prepared by fermenting sugar and tea extract with probiotic microorganisms collectively named as Symbiotic Culture of Bacteria and Yeast (SCOBY). Recently, fruits or leaves juices have become a new, favourable alternative medium for kombucha making. The employment of various starting materials and starter cultures during fermentations might result in the creation of several variable metabolites that may have various bioactivities. Thus, this study was aimed to determine the secondary metabolite content, antioxidant activities, antimicrobial effect against selected food borne pathogens and anticancer activities of the kombucha juices of papaya pulp and leaves fermented using locally isolated kombucha cultures, namely: yeast (Dekkera bruxellensis) and acetic acid producing bacteria (Komagataeibacter rhaeticus). Tolerance of these selected cultures against low pH and different bile salt concentrations were evaluated. D. bruxellensis MFS1 showed tolerance to pH 2 and higher and significant tolerance to bile salt at 0.3 %, 0.5%, and 1%. In contrast, K. rhaeticus MFS1 was only tolerant to pH 3 and higher and exhibited poorer tolerance to bile salt. Then, the kombucha juices supernatant were subjected to the profiling of organic acids and phenolic compounds using UPLC-PDA; quantification of total phenolic content; antioxidant activity measurement using FRAP, DPPH, and antimicrobial effect by well diffusion assay. The concentrations of acetic acid, L-malic acid, kojic acid and quinic acid increased in both fermented kombucha samples, with acetic acid being the most abundant form of organic acid being produced. Besides, chlorogenic acid, ellagic acid and 2,5-dihydroxybenzoic acid were also significantly increased after fermentation. In terms of antioxidant activities, papaya leaf kombucha (PLK) revealed higher activities of antioxidant compounds in the range of 89.56% compared to papaya pulp kombucha (PPK) (54.95%) DPPH inhibition. In addition, supernatant of fermented papaya leaves kombucha were able to inhibit the growth of pathogenic strains with higher zone of inhibition on most of the pathogens when compared to non-fermented and acetate samples. Other than that, anticancer properties of papaya-based kombucha were evaluated using in vitro and in vivo approaches. Using the MTT assay, higher concentrations of both kombucha pulp and leaves were shown to induce cellular growth suppression in two colon cancer cell lines, HT29 and SW 480. Both treated HT29 and SW480 shared a comparable cell population percentage pattern change from viable cells to early apoptotic, subsequently to the late apoptotic cell, as indicated by the Annexin V-FITC/PI assay. To determine how papaya kombucha administration affected the advancement of the cell cycles in HT29 and SW480, cell cycle analysis utilising flow cytometry was performed. Conclusively, kombucha treatment from PPK and PLK successfully inhibited the transition of the HT29 and SW480 cancer cells in vitro. Next, an in vivo study model named the AOM/DSS-induced ICR mice model was employed to evaluate the effect of papaya kombucha on the progression of colon cancer. The colon adenocarcinoma tumourigenesis process was delayed when the mice were fed using a low dose (0.7mL/kg) of papaya-based kombucha twice a day. On the contrary, feeding the mice with a high dose (1.8mL/kg) of similar kombucha treatment two times a day hastened colon carcinogenesis. Overall, significant dysbiosis in gut microbiota was observed in the control and AOM/DSS-induced groups. As the most predominant gut microorganisms, Bacteroidetes and Firmicutes demonstrated fluctuating abundances in healthy and ill mice. For instance, Bacteroidetes proliferation was significantly repressed by the growing of colon adenocarcinoma. Combining treatment with kombucha juices of papaya pulp and leaves and increasing Firmicutes growth was shown to help restore the bacterial load of several genera to near control levels. Using both in vitro and in vivo study designs, papaya kombucha beverages were shown to possess promising anticancer effects. This is because it contains potentially advantageous probiotic microbes, phenolic compounds, and substantial organic acid. Moreover, the acidic nature of kombucha enables it to regulate the pH balance within the intestine, helping to establish a healthy and balanced gut environment. The aforementioned positive results demonstrate the prospective benefits of incorporating papaya kombucha into one's diet as a functional food

Evaluation of antioxidant, antimicrobial and potential anticancer activities of papaya-based kombucha beverages on colon cancer

Traditional kombucha is prepared by fermenting sugar and tea extract with probiotic microorganisms collectively named as Symbiotic Culture of Bacteria and Yeast (SCOBY). Recently, fruits or leaves juices have become a new, favourable alternative medium for kombucha making. The employment of various starting materials and starter cultures during fermentations might result in the creation of several variable metabolites that may have various bioactivities. Thus, this study was aimed to determine the secondary metabolite content, antioxidant activities, antimicrobial effect against selected food borne pathogens and anticancer activities of the kombucha juices of papaya pulp and leaves fermented using locally isolated kombucha cultures, namely: yeast (Dekkera bruxellensis) and acetic acid producing bacteria (Komagataeibacter rhaeticus). Tolerance of these selected cultures against low pH and different bile salt concentrations were evaluated. D. bruxellensis MFS1 showed tolerance to pH 2 and higher and significant tolerance to bile salt at 0.3 %, 0.5%, and 1%. In contrast, K. rhaeticus MFS1 was only tolerant to pH 3 and higher and exhibited poorer tolerance to bile salt. Then, the kombucha juices supernatant were subjected to the profiling of organic acids and phenolic compounds using UPLC-PDA; quantification of total phenolic content; antioxidant activity measurement using FRAP, DPPH, and antimicrobial effect by well diffusion assay. The concentrations of acetic acid, L-malic acid, kojic acid and quinic acid increased in both fermented kombucha samples, with acetic acid being the most abundant form of organic acid being produced. Besides, chlorogenic acid, ellagic acid and 2,5-dihydroxybenzoic acid were also significantly increased after fermentation. In terms of antioxidant activities, papaya leaf kombucha (PLK) revealed higher activities of antioxidant compounds in the range of 89.56% compared to papaya pulp kombucha (PPK) (54.95%) DPPH inhibition. In addition, supernatant of fermented papaya leaves kombucha were able to inhibit the growth of pathogenic strains with higher zone of inhibition on most of the pathogens when compared to non-fermented and acetate samples. Other than that, anticancer properties of papaya-based kombucha were evaluated using in vitro and in vivo approaches. Using the MTT assay, higher concentrations of both kombucha pulp and leaves were shown to induce cellular growth suppression in two colon cancer cell lines, HT29 and SW 480. Both treated HT29 and SW480 shared a comparable cell population percentage pattern change from viable cells to early apoptotic, subsequently to the late apoptotic cell, as indicated by the Annexin V-FITC/PI assay. To determine how papaya kombucha administration affected the advancement of the cell cycles in HT29 and SW480, cell cycle analysis utilising flow cytometry was performed. Conclusively, kombucha treatment from PPK and PLK successfully inhibited the transition of the HT29 and SW480 cancer cells in vitro. Next, an in vivo study model named the AOM/DSS-induced ICR mice model was employed to evaluate the effect of papaya kombucha on the progression of colon cancer. The colon adenocarcinoma tumourigenesis process was delayed when the mice were fed using a low dose (0.7mL/kg) of papaya-based kombucha twice a day. On the contrary, feeding the mice with a high dose (1.8mL/kg) of similar kombucha treatment two times a day hastened colon carcinogenesis. Overall, significant dysbiosis in gut microbiota was observed in the control and AOM/DSS-induced groups. As the most predominant gut microorganisms, Bacteroidetes and Firmicutes demonstrated fluctuating abundances in healthy and ill mice. For instance, Bacteroidetes proliferation was significantly repressed by the growing of colon adenocarcinoma. Combining treatment with kombucha juices of papaya pulp and leaves and increasing Firmicutes growth was shown to help restore the bacterial load of several genera to near control levels. Using both in vitro and in vivo study designs, papaya kombucha beverages were shown to possess promising anticancer effects. This is because it contains potentially advantageous probiotic microbes, phenolic compounds, and substantial organic acid. Moreover, the acidic nature of kombucha enables it to regulate the pH balance within the intestine, helping to establish a healthy and balanced gut environment. The aforementioned positive results demonstrate the prospective benefits of incorporating papaya kombucha into one's diet as a functional food

Phytochemical composition and in vitro antioxidant activities of Citrus sinensis peel extracts

Background Citrus sinensis peels are usually discarded as wastes; however, they are rich sources of Vitamin C, fibre, and many nutrients, including phenolics and flavonoids which are also good antioxidant agents. This study aimed to examine phytochemical composition and antioxidant capabilities of C. sinensis peel extracted conventionally with different methanol/water, ethanol/water, and acetone/water solvents. Methods C. sinensis peels were subjected to extraction with 100%, 70% and 50% of methanol, ethanol, and acetone, respectively, as well as hot water extraction. Antioxidant activities of the peel extracts were examined via the 2,2-diphenylpicrylhydrazyl (DPPH) free radical scavenging activity, ferric reducing antioxidant power (FRAP) assay, and oxygen radical absorbance capacity (ORAC) assay. Total phenolic content and total flavonoid content of the extracts were measured via the Folin-Ciocalteau method and the aluminium chloride colorimetric method, respectively. Phenolic acid and organic acid composition of the peel extracts were further determined via high performance liquid chromatography (HPLC) while flavonoid content was identified via ultra performance liquid chromatography (UPLC). Results DPPH radical scavenging activity of C. sinensis peel extracts varied from 8.35 to 18.20 mg TE/g, FRAP ranged from 95.00 to 296.61 mmol Fe(II)/g, while ORAC value ranged from 0.31 to 0.92 mol TE/g. Significant level of association between the assays was observed especially between TPC and FRAP (R-square = 0.95, P < 0.0001). TPC of various C. sinensis peel extracts ranged from 12.08 to 38.24 mg GAE/g, with 70% acetone/water extract (AEC) showing the highest TPC. TFC ranged from 1.90 to 5.51 mg CE/g. Extraction yield ranged from 0.33 to 0.54 g/g DW and tended to increase with increasing water concentration in the solvent. In the phytochemical investigation, five phenolic acids were identified using HPLC, including gallic acid, protocatechuic acid, 4-hydroxybenzoic acid, caffeic acid and ferulic acid. A total of five organic acids including lactic acid, citric acid, L-mallic acid, kojic acid and ascorbic acid were quantified via HPLC. In addition, concentrations of six flavonoids including catechin, epigallocatechin, vitexin, rutin, luteolin and apigenin were determined via UPLC. Discussion and Conclusion Phytochemicals including phenolics and flavonoids in C. sinensis peel extracts exhibited good antioxidant properties. Among the extracts, 70% AEC with highest TPC and high TFC content showed greatest antioxidant activity in all three assays. Different phenolic acids, organic acids and flavonoids were also identified from the extracts. This study indicated that C. sinensis peels contained potential antioxidant compounds which could be exploited as value added products in the food industry

Short Communication Cloning of a Partial β-glucosidase Gene From Aspergillus terreus SUK-1

Abstract The breakdown of cellulose to glucose is catalysed by a complex cellulase enzyme which is comprised of three classes of enzymes; endoglucanase, exoglucanase and β-glucosidase. Many studies have been done on cellulase and several genes encoding for β-glucosidase have been reported. Here we describe the cloning and sequence analysis of partial putative β-glucosidase gene from Aspergillus terreus SUK-1. Its potential to produce cellulase enzyme has been studied and reported. A pair of specific primer was designed from the conserved regions of several fungal sequences taken out from the GenBank. PCR amplification has successfully amplified a DNA fragment of approximately 233 bp and this fragment was cloned (designated as SA3). The sequence analysis with BLAST showed high similarity towards β-glucosidase gene and protein. 80% and 79% homology were observed between SA3 nucleotide sequence and β-glucosidase gene of A. niger and both A. kawachii and A. aculeatus. Whereas, amino acid sequence of SA3 showed 83% homology towards β-glucosidase of A. niger, A. kawachii and A. aculeatus

COCONUT WATER VINEGAR: NEW ALTERNATIVE WITH IMPROVED PROCESSING TECHNIQUE

Vinegar is a condiment made from various sugary and starchy materials by alcoholic and subsequent acetic fermentation. Vinegar can be produced via different methods and from various types of raw material. A new alternative substrate for vinegar production namely mature coconut water has been tested and was compared with 2 common substrates which were coconut sap and pineapple juice. Substrates such as sap and juices have been found to have high amount of total soluble solids which corresponding to high sugar content in the substrates which is more than 14oBrix. Therefore, both substrates could be directly used for vinegar production without requirement of other carbon sources. However, coconut water which showed low Brix value need to be adjusted to 14oBrix by adding sucrose prior to the fermentation process. Substrates fermented with Saccharomyces cerevisiae have yielded 7-8% of alcohol within 7-10 days aerobic incubation at room temperature. The alcoholic medium were then used as a seed broth for acetic fermentation with Acetobactor aceti as inoculums and fermented for approximately 2 months to obtain at least 4% of acetic acid. Investigation on the effect of inoculum sizes and implementation of back-slopping technique were performed to improve the processing method for coconut water vinegar production. The results show that 10% of inoculum size was the best for acetic acid fermentation and the back-slopping technique has helped to reduce the process time of coconut water vinegar production

Cosmeceutical potentials and bioactive compounds of rice bran fermented with single and mix culture of Aspergillus oryzae and Rhizopus oryzae

In the present study, rice bran, one of the most abundant agricultural by-products in Malaysia, was fermented with single and mixed cultures of Aspergillus oryzae and Rhizopus oryzae. The fermented rice bran extracts were tested for their functional properties and compared to the non-fermented counterparts. Antioxidant activities as well as phenolics and organic acid contents were evaluated. Skincare-related functionalities were also tested by evaluating tyrosinase and elastase inhibition activities. Tyrosinase inhibition activity, measured to determine the anti-pigmentation effect of extracts, was found to be the highest in the extract of rice bran fermented with A. oryzae (56.18%) compared to other extracts. In determining the anti-aging effect of fermented rice bran extracts, the same extract showed the highest elastase inhibition activity with a value of 60.52%. Antioxidant activities were found to be highest in the mix-cultured rice bran extract. The results of phenolic and organic acid content were varied; the major phenolic acid detected was ferulic acid with a value of 43.19 μg/ml in the mix-cultured rice bran extract. On the other hand, citric acid was the major organic acid detected, with the highest content found in the same extract (214.6 mg/g). The results of this study suggest that the fermented rice bran extracts may have the potential to be further exploited as ingredients in cosmetics as well as in antioxidant-rich products

Screening of Potential Tannase-producing Fungi from Local Agri-industrial By-products using a Plate Assay and Submerged Fermentation

Tannase (Tannin Acyl Hydrolase EC 3.1.1.20) is an industrial inducible enzyme capable of hydrolyzing hydrolyzable tannin ester linkage gallotannin and ellagitannin, producing gallic acid and glucose. Tannase is extensively used in the pharmaceutical, chemical, cosmetics, textile, leather, food, feed, and beverage industries. In the beverage industry, tannase is used as a clarifying agent to clarify tannin present in coffee, coffee-flavored soft drinks, tea, and fruit juices by removing phenolic compounds. In the pharmaceutical industry, tannase is used to produce gallic acid, an intermediary compound in the production of antibacterial drug, trimethoprim, while in the food industry, tannase is used to synthesize crucial antioxidant food preservative propyl gallate (3,4,5-trihydroxybenzoate). Most of the tannase production utilizes bacteria such as Bacillus sp. as tannase producer under submerged fermentation, SmF. Despite the immense industrial potential of tannase, it has not fully been exploited due to lack of knowledge, and fewer studies reported filamentous fungi for tannase production. This study aimed to screen potential tannase-producing fungi from various agri-industrial by-products such as rice by-products, spent tea, spent coffee ground, banana peels, mango peels, desiccated coconut residue, soybean residue, sweet potato peels, and onions. Fungal isolate, J1 (Aspergillus niger) was identified as the efficient tannase-producing fungus due to the hydrolytic zone's largest diameter (60.7 ± 0.6) mm. It achieved high tannase activity with (6.86 ± 0.04) U/ml in submerged fermentation, SmF. In conclusion, filamentous fungi isolated from agri-industrial by-products have huge potential as an efficient tannase producer