Original Article


Background and Objective: Pseudomonas spp. are bacteria with the widest effects on food spoilage. These bacteria can be found in several environments such as soil and water. The major purpose of this study was to develop a software; by which, the growth behaviours of Pseudomonas spp. in culture media could be predicted.

Material and Methods: A total number of 509 bacterial data points of Pseudomonas spp. in culture media were collected from the ComBase database. Temperature and pH were used as the major prediction variables for the description of Pseudomonas spp. behaviours in culture media. Modified Gompertz, Baranyi and Huang models, the most commonly used models in predictive food microbiology to predict the count of microorganisms, were used as well. Fitting capability of each model was assessed and compared with other capabilities considering their statistical indices of the root mean square error, RMSE; coefficient of determination, R2; corrected Akaike information criterion, AICc; and Bayesian information criterion, BIC.

Results and Conclusion: Huang model provided better predictions with 0.951 of R2 and 0.825 of RMSE, compared to those of traditionally used models. Prediction capability of the Huang model was assessed considering externally collected data from the ComBase database. Huang model in the validation process provided satisfactory statistical indices (bias factor = 1.027 and accuracy factor = 1.075). These results have revealed that Huang model can be reliably used as a model of describing the growth behaviours of Pseudomonas spp. Furthermore, developed software in this study includes significant potentials for predicting Pseudomonas counts in culture media.

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

Isolation, purification and biochemical characterization of alkaline α-amylase from Bacillus subtilis strain W3SFR5 isolated from kitchen Wastes

Samuel Niyomukiza, Willis Owino, Julius Maina Mathara, Naomi Maina

Applied Food Biotechnology, Vol. 10 No. 1 (2023), 3 January 2023, Page 9-19

Background and Objective: Amylase is a hydrolytic enzyme that breaks starch into simple sugars. This enzyme includes uses in starch production, brewery, detergent formulation, paper production and pharmaceuticals as a digest aid. The aim of the present study was to isolate, identify and characterize an alkaline amylase from bacteria of food wastes.

Material and Methods: Bacteria were isolated using serial dilution, screened on agar plates and characterized through biochemical assessments and 16S rRNA sequencing. After optimizing the bacterial growth conditions using one factor at a time method, the alkaline amylase was extracted from the culture broth and partially purified using Sephadex G-75 chromatography. Enzyme activity generated by submerged fermentation was assessed using 3,5-dinitrosalicylic and recorded as the mean of three replicates.

Results and Conclusion: The bacterial isolate W3SFR5 showed high amylolytic activity in agar culture. Biochemical analysis and sequencing of the 16S rRNA verified the bacterial isolate as Bacillus subtilis (GenBank accession number: OM258620). Bacillus subtilis W3SFR5 was propagated within 30–50 ℃ and pH 6-9. The partially purified Bacillus subtilis W3SFR5 amylase included a molecular weight of 65 kDa and demonstrated a maximum specific activity of 216.02U mg-1. The optimum temperature for the enzyme was 60 °C and the pH was 9. The W3SFR5 amylase was actively stable under temperatures of 50–70 °C and pH of 7-9. Furthermore, 5 mM Fe2+ increased W3SFR5 amylase activity. The enzyme was more resistant to organic solvents, surfactants, inhibitors and oxidizing agents than that most amylases were. Additionally, results showed that W3SFR5 amylase was compatible with most commercial detergents, indicating that it could be used as a detergent additive.

Characterization of Plantaricin Genes and Lactic Acid Production by Lactiplantibacillus plantarum strains Isolated from Ishizuchi-Kurocha

Yolani Syaputri, Jiang Lei, Takema Hasegawa; Syiffa Fauzia; Nining Ratningsih, Tatang Suharman Erawan , Hitoshi Iwahashi

Applied Food Biotechnology, Vol. 10 No. 1 (2023), 3 January 2023, Page 21-31

Background and Objective: Ishizuchi-kurocha is a post-fermented tea that involved two main kinds of microorganisms, namely fungi and lactic acid bacteria, which are primary and secondary fermentation, respectively. Therefore, this research aimed to confirm the role of Lactiplantibacillus plantarum during secondary fermentation of Ishizuchi-kurocha and the anti-bacterial effect due to lactic acid production and genes detection of plantaricin.

Material and Methods: Antimicrobial were estimated using well diffusion method. Lactic acid was determined with spectrophotometric method. Detection of plantaricin genes were confirmed by Real-Time qPCR. The genes were sequenced through DNA Sequencing Analytical service by the Division of Genomic Research, Gifu University using the Multi-capillary DNA Sequencer ABI Prism 3100/3130 Genetic Analyzer and the data analyzed by the CLC Sequence Viewer 8.0 and BioEdit 7.2. Statistical analysis was evaluated by one-way of variance followed Tukey’s post hoc test using RStudio version 4.1.3.

Results and Conclusion: L. plantarum strain IYO1511 has higher antibacterial activities than strain IYO1501. In addition, L. plantarum strain IYO1511 produced higher lactic acid than strain IYO1501 and has plantaricin genes, plnA, plnEF, plnN, plnJ and plnK. However, L. plantarum strain IYO1501 only encoded plnEF, plnN, and plnJ. The plantaricin genes from the strains IYO1501 and IYO1511 were sequenced to identify the heterologous gene clusters of each species. It was discovered that plnA, plnEF, and plnJ of L. plantarum IYO1511 showed 100% similarity homology toward GenBank. The plnN of strain IYO1511 and plnEF of IYO1501 present extra base pairs inserted into the DNA. L. plantarum strains can be used as food preservative for artificial fermentation to control the safety and quality of the product of Ishizuchi-kurocha.The lactic acid and plantaricin were expected to inhibit pathogenically and spoilage bacteria to produce a unique acidic flavor as well as fragrance to Ishizuchi‐kurocha.

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

Probiotic Characteristics of Pediococcus pentosaceus and Apilactobacillus kunkeei Strains: The lactic Acid Bacteria Isolated from Bangladeshi Natural Honey

Jannatul Ferdouse, Shanta Paul, Tasneem Chowdhury, Ferdausi Ali, Saiful Islam, Tanim Jabid Hossain

Applied Food Biotechnology, Vol. 10 No. 1 (2023), 3 January 2023, Page 33-45

Background and Objective: Lactic acid bacteria are known for their strong probiotic effects on the hosts. The probiotic characterization of lactic acid bacteria from Bangladeshi natural honey is limited. The objectives of this study included isolation and assessment of the probiotic and safety characteristics of lactic acid bacteria in Bangladeshi honey.

Materials and Methods: Spread and streak plate techniques were used for the bacterial isolation and purification. Isolates were identified using 16S rRNA gene sequence analysis. Agar well diffusion and poisoned food methods were used for antibacterial and antifungal assessments, respectively. Antioxidant activity was carried out based on the microbial free-radical scavenging ability. Microbial autoaggregation, coaggregation and adhesion were assessed using cell sedimentation assay. Blood-agar was used in the hemolytic assay. Antibiotic susceptibility assay was carried out using disc diffusion method.

Results and Conclusion: From a total of 25 strains isolated from honey, ten Gram-positive, catalase-negative non-spore-forming isolates were selected and used in agar well diffusion assay. Three of the isolates showed prominent antimicrobial effects with large inhibition zones against all the pathogenic strains, including Bacillus cereus, Staphylococcus aureus, Vibrio cholerae, Salmonella typhi and Candida albicans. Extensive characterization of these isolates was carried out, which revealed their growth and biochemical characteristics and carbohydrate fermentation abilities. Moreover, 16S rRNA gene sequence analysis suggested that the isolates belonged to Pediococcus pentosaceus (two strains) and Apilactobacillus kunkeei. Their tolerance to simulated gastric conditions was assessed in vitro wherein the isolates showed significant survival in low pH, bile salts and phenol. They showed good adhesion ability to ethyl acetate, chloroform and xylene as well as high autoaggregation and coaggregation characteristics. Moreover, free-radical scavenging activity of the isolates suggested the presence of considerable antioxidant effects. In safety assessment, the isolates did not show hemolytic activities and were resistant to several antibiotics. Therefore, these results indicate that honey can be an important source of beneficial lactic acid bacteria species providing several probiotic advantages.

Conflict of interest: The authors declare no conflict of interest

Increasing Protein Content of Tomato Pomace using Solid-State Fermentation with Industrial Bakery Yeasts

Behnaz Shakouri, Valiollah Babaeipour, Mansour Mashreghi

Applied Food Biotechnology, Vol. 10 No. 1 (2023), 3 January 2023, Page 47-59

Background and Objective: Tomato pomace as the major waste of tomato paste can be used in food formulation due to its nutritional and technological characteristics. The aim of this study was to create a cheap simple method to increase the protein content of tomato pomace, which could be used as a cheap and more efficient food source for livestock and poultry.

Material and Methods: The study was carried out in three stages: 1) selection of further appropriate yeasts by assessing effects of two types of avaiable Saccharomyces cerevisiae and Saccharomyces bulardi industrial yeasts and fermentation time in the moisture content of  5% tomato pomace with particle size of less than 500 μm on the protein content of tomato pomace, 2) asessment of optimum conditions for increasing the protein content of tomato pomace by assessing effects of four parameters of the quantity of the yeasts, initial moisture content, substrate particle size and cultivation time in three levels, based on the Taguchi method in nine experiments in laboratory scale, 3) increase of the protein content of tomato pomace in bench scale tray bioreactor by investigation of three factor substrate depth, distance between trays and substrate particle size in three levels and one aeration factor in two levels based on the Taguchi method.  

Results and Conclusion: Saccharomyces cerevisiae and 5-d cultivation time were chosen to continue the study. Under optimal conditions in laboratory scale, 0.03 g dry yeast/g tomato pomace of yeast, moisture content of 70% (w w-1), particle size of less than 150–250 μm (mesh 100) and process time of 5 d, protein content of 24.72% with fat content of 3.29%, ash of 16.45% and carbohydrate of 55.52% (w w-1) were achieved. Under optimal conditions, including bed depth of 1.2 cm, tray distance of 4 cm, particle size of 250-500 μm (mesh 60) without aeration, the maximum protein content of 25.82% were achieved, which were more than 80%, compared to the primary tomato pomace protein content (14.21% w w-1). This is the highest protein content already reported for tomato pomace, using the simplest technology at the lowest cost.

Conflict of interest: The authors declare no conflict of interest

Technological Assessment of Autochthonous Lactic Acid Bacteria and their Antibacterial Activities Against Food borne Pathogens in Goat Milk Lactic Cheese

Fahimeh Assari, Naheed Mojgani, Mohammad Reza Sanjabi, Saeed Mirdamadi, Hoda Jahandar

Applied Food Biotechnology, Vol. 10 No. 1 (2023), 3 January 2023, Page 61-71

Background and Objective: Lactic cheese is a highly consumed dairy product that is of great nutritional values. However, great concerns are reported regarding their safety, microbial quality and short shelf life. Addition of antimicrobial compound-producing lactic acid bacteria as protective cultures is known to improve safety and ensure quality of food products. The current study was carried out to assess antibacterial and several technological characteristics of autochthonous Lactic acid bacterial isolates for their use in goat milk lactic cheese to control Staphylococcus aureus and Listeria monocytogenes.

Material and Methods: Antibacterial and other technological characteristics, including proteolytic activity, diacetyl production, autolytic activity and survival, in various NaCl concentrations and at suboptimal temperatures of several Lactic acid bacteriasolates were assessed. The potent isolates were identified to species level by phenotypic and genotypic methods (16 srRNA gene sequencing) and finally they were assessed for their ability to inhibit Staphylococcus aureus and Listeria monocytogenes in prepared goat milk lactic cheese.

Results and Conclusion: Lactic acid bacterial isolates, showing significant antibacterial action, proteolytic activity and diacetyl production, were identified as Lactiplantibacillus plantarum RTCC 1290-1, Lactobacillus acidophilus RTCC 1299 and Lacticaseibacillus casei RTCC 1296-1. These isolates survived at 25 and 45 ºC, tolerated up to 8% NaCl and 55 ºC for 10 min (p≤0.01). Lactobacillus acidophilus demonstrated the highest autolytic activity (38.69%) followed by Lacticaseibacillus casei (28.44%) and Lactiplantibacillus plantarum (18.38%), respectively. Cocultures of the selected lactic acid bacterial isolates in goat milk lactic cheese resulted in a 5 log CFU g-1 decrease in Listeria monocytogenes, while, Staphylococcus aureus counts decreased by only 3 Log CFU g-1 (p≤0.05) during a month of storage at room temperatures. However, lactic acid bacterial counts increased 2–4 Log CFU g-1 from Day 15, which was reported stable up to Day 30. In conclusions, Lacticaseibacillus casei, Lactiplantibacillus plantarum and Lactobacillus acidophilus bears promising biopreservative effect for the control of foodborne pathogens.

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

Background and Objective:

Chlorella sp. is one of the most studied microalgae because it contains compounds with pharmacological properties, critical to human health. As diversity of the biomolecule changes with the growth of microalgae, it is essential to assess these changes during various growth phases for the targeted extraction of biomolecules. In this study, changes in biomolecules extracted from Chlorella sp. at various growth phases were analyzed using various solvent systems.

Material and Methods:

Chlorella sp. was isolated from a freshwater pond and three solvents (ethanol, methanol and acetone) were used to extract phyto-compounds from biomasses from three different growth phases (exponential, early stationary and late stationary phases). Analysis of the fatty acids, total phenolic and flavonoid levels, and antioxidant activity were all performed on extracts from different growth phases.

Results and Conclusion:

Total flavonoid concentration was highest during the late stationary growth phase in contrary to the early growth stages. In contrast, total phenolic content was highest in the exponential phase. Fatty acid profile revealed that the early growth phases of the algae included higher levels of omega-3 and omega-6 polyunsaturated fatty acids in comparison to the late stationary phase. The extract phyto-constituents varied in quantity and type during the microbial growth and were dependent on the solvent system. Correlations between the total phenolic content, fatty acids and DPPH free radical scavenging were positive, demonstrating that the quantity and type of phyto-constituents varied depending on the stages of algal growth. Additional investigations of Chlorella sp. can develop its potentials for commercial uses.

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