Original Article

Effects of Postbiotics from Food Probiotic and Protective Cultures on Proliferation and Apoptosis in HCT-116 Colorectal Cancer Cells

Behnam Omidi sarajar, Arash Alizadeh, Mehran Moradi, Vahid Shafiei Irannejad

Applied Food Biotechnology, Vol. 10 No. 2 (2023), 18 March 2023, Page 85-101

Background and Objective: Postbiotics are microbial-derived soluble products, which are released during the growth and fermentation process of beneficial microorganisms in gastrointestinal tract, food and complex microbiological culture systems (cell-free supernatant or extracellular extract) or after cell lysis (intracellular extract). Colorectal cancer is one of the most common cancers within the leading causes of cancer mortality worldwide, which can be associated with a defeated gastrointestinal barrier. In this study, potential functionality of the extracellular and intracellular extracts of probiotics (Latilactobacillus sakei, LS) and protective culture (FreshQ®, FQ) on proliferation and cell survival of HCT-116 colon cancer epithelial cells was investigated.

Material and Methods: Probiotic bacteria were cultivated in de Man, Rogosa and Sharpe broth and then postbiotics was isolated by centrifugation and sonication. The achieved solutions were lyophilized and stored until use. Moreover, HCT-116 cells were exposed to various concentrations of Latilactobacillus sakei and FreshQ® extracts (1.25-40 mg ml-1) for 24 h and then effects of these products on cell cytotoxicity, proliferation and apoptosis were investigated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, wound healing and AO/EB assays.

Results and Conclusion: Extracellular and intracellular extracts of Latilactobacillus sakei and FreshQ the decreased in cell viability based on the postbiotic concentrations (p≤0.05), while cell proliferation was inhibited by extracellular and intracellular extracts of Latilactobacillus sakei and FreshQ® in wound healing assay. Results showed that postbiotics could induce apoptosis evidenced by acridine orange/ethidium bromide staining. In summary, Latilactobacillus sakei and FreshQ® postbiotics are able to decrease cell viability and proliferation and enhance apoptosis in HCT-116 colorectal cancer cells. In addition, FreshQ® postbiotics seemed more potent than that Latilactobacillus sakei postbiotics did.

Conflict of interest: The authors declare no conflict of interest.

Production of Bioactive Peptides in Milk Using Two Native Strains of Levilactobacillus brevis

Leila Yousefi, Mohammad Bagher Habibi Najafi, Mohammad Reza Edalatian Dovom

Applied Food Biotechnology, Vol. 10 No. 2 (2023), 18 March 2023, Page 103-111

Background and Objective: Milk proteins are precursors of several biologically active peptides. One of the methods of producing these peptides is fermentation using lactic acid bacteria. The aim of this study was to investigate production of antioxidant and angiotensin-I converting enzyme inhibitory bioactive peptides in cow milk fermented by two strains of Levilactobacillus brevis.

Material and Methods: Two strains of Levilactobacillus brevis KX572376 (M2) and Levilactobacillus brevis KX572382 (M8) were used in fermentation of low-fat cow milk. Moreover, pH changes, proteolytic activity, water-soluble extract biological activity (antioxidant activity and angiotensin-I converting enzyme inhibition) of the samples and peptide fraction less than 3 kDa were investigated at 24 and 48 h of fermentation (30 °C). Peptide profile of the superior sample was analyzed as well. Statistical analysis was carried out using one-way of variance, Tukey test and SPSS software v.25.

Results and Conclusion: The two strains decreased milk pH to a similar level in the first 24 h. Quantities of free amine groups in the samples treated with M2 and M8 strains within 24 and 48 h of fermentation were significantly different (p≤0.05), compared to the control sample. In the first 24 h of fermentation, no difference was observed in the quantity of free amines of M2 and M8 samples. In the second 24 h, further free amine groups were produced due to the activity of M8 strain in milk. Antioxidant activity of the water-soluble extracts of M2 and M8 samples was significantly (p≤0.05) higher than that of the control sample during fermentation. Antioxidant activity in fractions less than 3 kDa did not show significant differences in M2 and M8 samples at 24 and 48 h of fermentation. In the control sample, no antioxidant activity was observed in fractions less than 3 kDa. The highest ACE inhibitory activity in fractions less than 3 kDa of M8 was observed after 48 h. No angiotensin-I converting enzyme inhibition was seen in fractions less than 3 kDa of M2 and control sample. The RP-HPLC peptide patterns of the fraction less than 3 kDa of M8 and control sample were different, which was a justification for the biological activity in this sample.

Conflict of interest: The authors declare no conflict of interest.

Biopolymeric Nanoparticles, Pickering Nanoemulsions and Nanophytosomes for Loading of Zataria multiflora Essential Oil as a Biopreservative

Mansoureh Taghizadeh, Seid Mahdi Jafari, Kianoush Khosravi Darani, Mahmood Alizadeh sani, Saeedeh Shojaee Aliabadi , Nader Karimian Khosroshahi, Hedayat Hosseini

Applied Food Biotechnology, Vol. 10 No. 2 (2023), 18 March 2023, Page 113-127

Background and Objective: Essential oils include low solubility, poor bioavailability and rapid release, which may limit their use as bioactive compounds in foods and medicine. Nanoencapsulation can preserve inherent qualities of essential oils and improve their physicochemical characteristics and health benefits. Focus of the present study was on the loading of essential oils from Zataria multiflora in pickering nanoemulsions, nanoparticles and nanophytosome. In addition, the present study assessed how these systems affected their physicochemical characteristics and antioxidant and antimicrobial activities, compared to free-essential oils.

Material and Methods: Encapsulation of Zataria multiflora Boiss essential oil in nanocarriers as a novel phytoconstituents delivery system was carried out using three various methods. Physicochemical characterization of nanocarriers was studied using dynamic light scattering, Fourier transform infrared spectroscopy, field emission scanning electron microscope, confocal laser scanning microscopy, optical microscope and antioxidant activity. The minimum inhibitory and bactericidal concentration assessment effects against Listeria monocytogenes at 24 h and temperatures (10, 25 and 37 °C) were investigated. Encapsulated Zataria multiflora Boiss essential oil with subinhibitory concentrations (0.25, 0.5 and 0.75) in hamburger formulation was selected as a food model for chemical, microbiological and sensory evaluation.

Results and Conclusion: In general, this study compared three types of biocarriers with free essential oils. Primarily, nanophytosome showed promising results in delaying oxidation and in antimicrobial and sensory assessments, compared to two other nanocarriers. In conclusion, essential oil nanophytosomes of Zataria multiflora Boiss include the potential as an efficient natural food preservative.

Conflict of interest: The authors declare no conflict of interest.

Antibacterial activities of purified monolaurin obtained from enzymatic glycerolysis of palm kernel olein-stearin blend against Bacillus subtilis

Ngatirah Ngatirah, Chusnul Hidayat, Endang Sutriswati Rahayu, Tyas Utami

Applied Food Biotechnology, Vol. 10 No. 2 (2023), 18 March 2023, Page 129-140

Background and Objective: Monolaurin can be produced by enzymatic glycerolysis of a mixture of palm kernel olein-stearin (PKOo-PKS). Monolaurin has the ability as an antibacterial agent against both gram-positive and negative bacteria. Limited studies investigated the activity against bacteria of monolaurin on B. subtilis cell and spores. Therefore, this research aimed to study the effect of a purified monolaurin obtained from enzymatic glycerolysis of PKOo-PKS blend on growth, spore germination, cell surface hydrophobicity, and cell structure of B. subtilis FNCC 0060.

Material and Methods: Monolaurin was produced using palm kernel-olein blend material under the best enzymatic glycerolysis conditions and purified using solvent (hydroalcoholic) method. The effect of a purified monolaurin on B. subtilis growth and spore germination was studied using nutrient broth. Analysis of cell surface hydrophobicity (CSH) was carried out using MATHS (microbial adhesion to hydrocarbons) assay, while the cell structure was observed using Transmission Electron Microscope (TEM). As an experimental design, the completely randomized design (CRD) was used, and each treatment was triplicated. Analysis of variance (ANOVA) and Duncan multiple range tests (DMRT) were used to analyze the data, which revealed a significant difference.

Results and Conclusion: This result showed that purified monolaurin inhibited the growth of B. subtilis FNCC 0060. Meanwhile, the development of B. subtilis in nutrient broth with monolaurin at 100-5,000 ppm was slower than without. A higher concentration in the medium decreases the specific growth rate and increases the doubling time. Spores of B. subtilis FNCC 0060 in nutrient broth with monolaurin at 100-1,000 ppm concentration were delayed in germination up to seven days of incubation. The presence of monolaurin significantly reduces the percent adherence of bacterial cells. Furthermore, the cytoplasm of B. subtilis FNCC 0060 appeared to have shrunk considerably, causing cell cytoplasm damage and disorganization of the components.

Formulation of Sausage with Encapsulated GO: Physicochemical, Microbial and Sensory Properties

Khadijeh Khoshtinat, Mohsen Barzegar, Mohammad Ali Sahari, Zohreh Hamidi

Applied Food Biotechnology, Vol. 10 No. 2 (2023), 18 March 2023, Page 141-154

Background and Objective: Consumers prefer not to use synthetic preservatives so, natural ones such as essential oils (in this research, garlic oil (GO) are mostly consumed in food products as an antimicrobial agents. Application of garlic oil in food industries is limited. Because of its water insolubility, volatility, pungent odour and flavor, and low stability. Encapsulation could eliminate its restrictions. In the present study, different concentrations of free and encapsulated garlic oil in ß-cyclodextrin (GO/ß-CD) were used for formulation of sausage

Material and Methods: Five types of sausage were produced, including control (without garlic oil), containing free garlic oil (GO), and three samples containing 546, 818, and 1364 mg of encapsulated garlic oil per kg. After that, changes of pH, color, oxidation indices (PV and TBARs(, monitoring of B. cereus, S. aureus, E. coli, and S. enterica, and sensory (taste, color, odor, and acceptability by 8-point hedonic test) properties were studied during 29 days storage at 4oC.

Results and Conclusion: By increasing the GO/ß-CD content of samples, the overall color changes decreased. The pH of all produced sausages remained constant. At the end of storage, PV was not significantly changed, and TBARs changing was very low. S. aureus was the most susceptible, followed by S. enterica, E. coli, and B. cereus. In sensory evaluation, the panelists did not distinguish between the control and the samples containing GO/ß-CD. Encapsulation of garlic oil with beta-cyclodextrin did not affect the taste of the produced sausages. Due to good antimicrobial properties of garlic oil, it can be used in meat products as a natural preservative.

Conflict of interest: The authors declare no conflict of interest.