Review Article

Lukanka, a Semi-Dried Fermented Traditional Bulgarian Sausage: Role of the Bacterial Cultures in its Technological, Safety and Beneficial Characteristics

Svetoslav Dimitrov Todorov, Clarizza May Dioso, Min-Tze Liong, Tonka Vasileva, Penka Moncheva, Iskra Vitanova Ivanova, Ilia Iliev

Applied Food Biotechnology, Vol. 9 No. 4 (2022), 1 October 2022, Page 261-273

Background: Production of different fermented meat products is a well-known practice done in different European countries since ancient times. Fermentation of primary materials and/or smoking and salting processes are part of the preservation processes and is important for the formation of final products which is inherent in South European countries. Originally, fermentation of meat products is intended for preservation and safe storage for long periods of time. However, nowadays, gastronomical properties of fermented meat products are essential in obtaining specific flavor, odor, color and structure of the sausages which consumers highly prefer. Emphasis is given on gastronomic characteristics, which results from the various combination of raw meat, specific spices and the natural microbiota or conducted fermentation processes by application of specific starter cultures. Seven bacterial genera (Lactobacillus, Leuconostoc, Staphylococcus, Enterococcus, Lactococcus, Micrococcus and Streptococcus) are commonly used as meat starter cultures. Complex fermentation processes that occur during the ripening of the fermented meat products are the results of the interaction between bacterial starter cultures, remaining enzymes in the muscle and fat tissue and available bacterial enzymes.

Objective: The present overview aims to provide information related to the characterization of the specific microbiota associated with lukanka, a naturally-fermented semi-dried Bulgarian sausage. What is the specificity of its fermentation processes; how do different starter and indigenous meat microbiota interfere to form specific final products; what is the role of starter and adjunct cultures in the safety of the products; how is the Bulgarian lukanka classified in the perspective of other Mediterranean dry fermented sausages? These are some of the questions that this review will discuss.

Original Article

Biosynthesis of Nano-Calcite and Nano-Hydroxyapatite by the Probiotic Bacteria of Bacillus subtilis and Bacillus coagulans

Sabere Nouri, Rasoul Roghanian, Giti Emtiazi, Rasoul Shafiei

Applied Food Biotechnology, Vol. 9 No. 4 (2022), 1 October 2022, Page 275-286

Background and objective: In recent years, the green synthesis of nanomaterials has received more attention than chemical synthesis due to its eco-friendly and compatibility. Material and methods: In this study Bacillus subtilis and Bacillus coagulans, two potential probiotics, were used and grown Bacillus in medium culture containing insoluble calcium phosphate produced nano-hydroxyapatite and nano-calcite. The nano-hydroxyapatite was purified from nano-calcite by heat treatment and washing with a 200 nm filter. The structures, characteristics, and elemental analysis of nano-sized material were studied by Fourier transform infrared spectroscopy, X-ray diffraction, scanning electron microscope, ultraviolet-visible spectroscopy, energy dispersive X-ray, and X-ray fluorescence. Results: The results showed that hydroxyapatite is made only in a medium containing insoluble calcium phosphate sublimated with urea which is induced phosphatase and urease. Here, for the first time, the braided bacterial nano-hydroxyapatite similar to the bone structure was made in the medium, which caused the production of urease and phosphatase (Maximum 99 U/L) enzymes, and the particle size was less than 100 nm. The ratio of calcium to phosphorus in crude hydroxyapatite and calcite crystal particles made by B. coagulans was 2.9, however, this ratio for pure hydroxyapatite was 1.7. Conclusion: Since the particles are made by antibacterial probiotics, the biological production of these particles makes them a suitable candidate to be used in food, toothpaste, and sanitation products. Braided hydroxyapatite can substitute the needle-like type of food additives for infants and elders due to its safety.

Optimization of Gamma Aminobutyric Acid Production from Various Protein Hydrolysates by Lactiplantibacillus plantarum MCM4

Ali Moayedi, Zahra Zareie, Forough Yaghoubi, Morteza Khomeiri

Applied Food Biotechnology, Vol. 9 No. 4 (2022), 1 October 2022, Page 287-296

Background and Objective: Gamma aminobutyric acid (GABA) is a non-protein amino acid with various physiological properties that is used as synthetic drugs for curing anxiety, acute stress, and hypertension. In the recent years, producing the GABA-enriched foods and supplements via biological methods has gained much attention.

Material and Methods: In this study, at first, one factor at a time (OFAT) approach was used to study the effect of various kinds of protein hydrolysates (soy protein isolate (SPI), whey protein concentration (WPC) and casein) at different concentrations, fermentation time (24 to 72h) and inoculum size (1, 3, and 5% v/v) on GABA synthesis. Then, the most effective parameters i.e., soy protein hydrolysate (SPH) concentration, inoculum size, and fermentation time were further employed by central composite design (CCD)-based response surface methodology (RSM) for GABA synthesis by Lactobacillus plantarum MCM4.

Results and Conclusion: Among different protein hydrolysates, SPH was found to be more suitable for GABA synthesis. Moreover, higher GABA content was obtained when soy protein with extended enzymatic hydrolysis (SPH6) was used as the substrate. The polynomial mathematic model could predict the GABA synthesis successfully. The optimization using CCD indicated that the maximum GABA syntehsis yield (19.387 mg GABA/100 mL) was achieved under optimium conditions (fermentaion time of 28.99 h, inoculum size of 3.65% v/v, and SPH6 concnetration of 3.89% w/v). Overall, L. plantarum MCM4 was found to be a novel LAB species to produce GABA from inexpensive sources.

Background and Objective: Lactic acid bacteria have recently become one of the major topics of discussion in fields of health, food industry, science, animal husbandry and agriculture. Lactic acid bacteria have been widely used in fermentation of various types of food products from animals, fish and plants that act as preservatives and include positive effects on human health and beauty. One source of lactic acid bacteria is Mystacoleucus padangensis fish from Singkarak Lake, West Sumatera, Indonesia, where a probiotic bacterium of Limosilactobacillus fermentum with antimicrobial potential is isolated. The aim of this study was to assess antimicrobial potential of Limosilactobacillus fermentum isolated from Bilih fish from Singkarak Lake, Indonesia.

Material and Methods: Methods of this study were as follows: isolation of lactic acid bacteria from Bilih fish from Singkarak Lake, followed by antimicrobial activity assessment of the raw bacteriocin supernatant. Then, 16S rRNA was used to assess species of the lactic acid bacteria isolates. From the three samples, one sample of Isolate IB1, with potential antimicrobial activity was reported.

Results and Conclusion: Results of the study showed that the morphological and biochemical characteristics of lactic acid bacteria included Gram-positive, bacilli form and catalase-negative belonged to the group of homofermentative bacteria. The greatest antimicrobial activities were shown by IB1 Escherichia coli 0157:H7 (27.29 mm), Staphylococcus aureus ATCC 25923 (14.17 mm) and Listeria momocytogenes CFSAN 004330 (11.44 mm) while the diameter of inhibition zone by the supernatant of Lactic acid bacteria Escherichia coli 0157:H7 crude bacteriocin was 16.89 mm while Staphylococcus aureus ATCC 25923 did not form inhibition zone. The observed antimicrobial activity was 17.19 mm for Listeria monocytogenes CFSAN 004330 at neutral pH. Results of the molecular identification using 16S rRNA showed that the isolated lactic acid bacteria included similarities with Limucilactbacillus fermentum strain 17059 16S, which included antimicrobial potentials against pathogenic bacteria. Limosilactobacillus fermentum 17059 16S can be used as an antidiarrheal and antityphoid agent in humans and as a natural preservative in foods.

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



Background and Objective: Disposal of fishery wastes is one of the environmental challenges. Converting wastes into valuable products is an economical solution to solve this environmental problem. One of the wastes of fishery products is shrimp shell waste, which contains large quantities of chitin. Chitin and its derivative, chitosan, include several uses in various industries, especially the food industry. The aim of the present study was to extract chitin from shrimp shell wastes using co-fermentation with mixed microbial cultures. Chitosan production by deacetylation of chitin using various concentrations of NaOH was another aim of this study.

Material and Methods: Batch fermentation was carried out to extract chitin from the shrimp shell using mixed cultures of two microorganisms, Lactiplantibacillus plantarum PTCC 1745 and Bacillus subtilis PTCC 1720. The Logistic model was used to assess the microbial growth rate. To prepare chitosan from chitin, deacetylation was carried out using NaOH solution. Morphological structures of the chitin and chitosan were studied using scanning electron microscopy. Functional groups of the synthesized chitin and chitosan were assessed using Fourier transform infrared spectroscopy.

Results and Conclusion: High levels of demineralization and deproteinization were achieved using co-fermentation with the mixed microbial populations at 60 g.l-1 of glucose concentration. Chitin was produced with high purity and the protein and ash contents included 1.43 and 1.26%, respectively. Data predicted by the Logistic model were fairly matched the experimental data. A maximum cell growth rate of 0.065 (h-1) was achieved at 60 g.l-1 of initial glucose concentration at 35 °C. The optimal value of deacetylation (88.2%) was achieved using 50% NaOH solution at 100 °C. The results showed that the use of mixed culture of acid-producing and proteolytic microorganisms is highly effective for extracting chitin from shrimp shell waste.

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

Synthesis, Characterization, and Evaluation of Anti-Helicobacter Activity of Chitosan and Pectin Microparticles Containing Zataria multiflora Extract In Vitro

Sanaz Malek, Mir Khalil Pirouzifard, Abbas Yadegar, Solmaz Mahdigholi Vand Bonab, Behrooz Jannat, Masoumeh Moslemi

Applied Food Biotechnology, Vol. 9 No. 4 (2022), 1 October 2022, Page 321-331

Background and Objective: Helicobacter pylori infection is of widespread diseases in the world. The most common treatment for remediation of its symptoms is administration of antibiotics, which are not efficient enough in some patients and resulted in antibiotic resistance. Given the adverse effects arising from antibiotics in clinical studies, we aimed to fabricate chitosan- and pectin-based micro-particles containing Zataria multiflora extract for suppression of Helicobacter pylori in the laboratory.

Materials and Methods: Chitosan and pectin micro-capsules alone and in combination with Zataria multiflora extract were prepared by spray dryer. The powders were further characterized by FT-IR, zeta sizer, and scanning electron microscope. Simulated gastric fluid was also prepared for evaluation of anti-Helicobacter potency of the samples.

Results and Conclusion: Chitosan 1% w v-1 + 0.5 ml Zataria multiflora extract (CE0.5), pectin 1% w v-1 + 0.3 ml Zataria multiflora extract (PE0.3), and pectin 1% w v-1 (P1) could significantly suppress the bacteria under simulated gastric condition. The least survivability of Helicobacter pylori was 45.4% and achieved for CE0.5, followed by 45.70% and 46.6% for PE0.3 and P1, respectively. Electrostatic charge of the biopolymers and phenol compounds of the extract greatly affected the integrity of bacterial cell wall. According to FT-IR spectra, Zataria multiflora extract was physically entrapped in chitosan and pectin layers, which helped its better antimicrobial activity in vitro. Regarding the significant anti-Helicobacter activity of our selected formula, they would be considered as complementary treatment for antibiotics in eradication of Helicobacter pylori infection. Although further in vivo experiments are required to validate the current findings.

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