Milk Fermentation Process Research Articles

Substitution of Asp29 with Asn29 in the metallochaperone UreE of Streptococcus thermophilus DSM 20617T increases the urease activity and anticipates urea hydrolysis during milk fermentation

Urease operon is highly conserved within the species Streptococcus thermophilus and urease-negative strains are rare in nature. S. thermophilus MIMO1, isolated from commercial yogurt, was previously characterized as urease-positive Ni-dependent strain. Beside a mutation in ureQ, coding for a nickel ABC transporter permease, the strain MIMO1 showed a mutation in ureE gene which code for a metallochaperone involved in Ni delivery to the urease catalytic site. The single base mutation in ureE determined a substitution of Asp29 with Asn29 in the metallochaperone in a conserved protein region not involved in the catalytic activity. With the aim to investigate the role Asp29vs Asn29 substitution in UreE on the urease activity of S. thermophilus, ureE gene of the reference strain DSM 20617T (ureEDSM20617) was replaced by ureE gene of strain MIMO1 (ureEMIMO1) to obtain the recombinant ES3. In-gel detection of urease activity revealed that the substitution of Asp29 with Asn29 in UreE resulted in a higher stability of the enzyme complexes. Moreover, the recombinant ES3 showed higher level of urease activity compared to the wildtype without any detectable increase in the expression level of ureC gene, thus highlighting the role of UreE not only in Ni assembly but also on the level of urease activity. During the growth in milk, the recombinant ES3 showed an anticipated urease activity compared to the wildtype, and analogous milk fermentation performance. The overall data obtained by comparing urease-positive and urease-negative strains/mutants confirmed that urease activity strongly impacts on the milk fermentation process and specifically on the yield of the homolactic fermentation.

Lactococcus lactis in Dairy Fermentation—Health-Promoting and Probiotic Properties

The use of lactic acid bacteria (LAB) in the fermentation process to produce fermented foods has a long history. Furthermore, LAB are beneficial microorganisms known for their health-promoting characteristics. During fermentation, LAB have the capacity to produce significant amounts of bioactive substances, such as peptides, bacteriocins, lactic acid, exopolysaccharides (EPSs), enzymes, and others. Lactococcus lactis as one of the best-known and well-characterized species of LAB serves as a model organism for studying LAB. For a very long time, L. lactis has been used in milk fermentation, both in well-monitored industrial settings and on a small scale in traditional operations. Furthermore, L. lactis is a vital microorganism in the dairy food fermentation industry due to its role in acidification, flavor development, and the creation of various dairy products, including cheese, fermented butter, and others. The novelty of this review is the comprehensive and organized presentation of the main benefits of the use of L. lactis in milk fermentation processes including technological and safety features relevant for the dairy industry, probiotic potential, the ability to produce bioactive compounds (e.g., bacteriocins, GABA), and the recent development of such bacteria research methods like whole genome sequencing (WGS).

Assessment of probiotic properties of lactic acid bacteria isolated from an artisanal Colombian cheese

Lactic Acid Bacteria play an important role in the milk fermentation processes of traditional cheeses and have become an important target for the development of novel cheese cultures because of their ability to confer health benefits. This study aimed to evaluate the probiotic potential of 12 Lactic Acid Bacteria (LAB) strains previously isolated and molecularly identified from an artisanal Colombian Double-Cream Cheese. Probiotic properties, including safety (hemolysis and sensibility to antibiotics), pH and bile salt tolerance, auto-aggregation, cell surface hydrophobicity, antibacterial activity, and exopolysaccharide production, were examined. None of the strains were hemolytic, and Pediococcus (16, 18) and Lactobacillus (28, 29) were found to be sensitive to all antibiotics. Moreover, all the strains tolerated pH (3.0, 6.5 and 8.0) and bile salt conditions (0.3, 0.6 and 1.0 % w/v). Pediococcus pentosaceus (16), Leuconostoc citreum (17), Pediococcus acidilactici (18), Enterococcus faecium (21,22), Enterococcus faecalis (24) and Limosilactobacillus fermentum (29) exhibited medium autoaggregation and affinity to chloroform. Six of the strains exhibited a ropy exopolysaccharide phenotype. Antibacterial activity against foodborne pathogens, Salmonella Typhimurium ATCC 14028, Listeria monocytogenes ATCC 19111, Escherichia coli ATCC 25922 and Staphylococcus aureus ATCC 25923, was found to be strain dependent, with the strains 16, 18, 21, 26, 28 and 29 presenting a higher inhibition (>4 mm) against all of them. According to Principal Component Analysis, P. pentosaceus (16), Leu. mesenteroides (26), L. casei (28), L. fermentum (29), and E. faecium (21) showed strong probiotic properties. Our findings suggest that five strains out of the 12 sampled strains are potential probiotics that could be used in the processing of traditional dairy products on an industrial scale to improve their quality.

The pH-dependent lactose metabolism of Lactobacillus delbrueckii subsp. bulgaricus: An integrative view through a mechanistic computational model.

The fermentation process of milk to yoghurt using Lactobacillus delbrueckii subsp. bulgaricus in co-culture with Streptococcus thermophilus is hallmarked by the breakdown of lactose to organic acids such as lactate. This leads to a substantial decrease in pH - both in the medium, as well as cytosolic. The latter impairs metabolic activities due to the pH-dependence of enzymes, which compromises microbial growth. To quantitatively elucidate the impact of the acidification on metabolism of L. bulgaricus in an integrated way, we have developed a proton-dependent computational model of lactose metabolism and casein degradation based on experimental data. The model accounts for the influence of pH on enzyme activities as well as cellular growth and proliferation of the bacterial population. We used a machine learning approach to quantify the cell volume throughout fermentation. Simulation results show a decrease in metabolic flux with acidification of the cytosol. Additionally, the validated model predicts a similar metabolic behaviour within a wide range of non-limiting substrate concentrations. This computational model provides a deeper understanding of the intricate relationships between metabolic activity and acidification and paves the way for further optimization of yoghurt production under industrial settings.

Bifidobacterium adolescentis B8589- and Lacticaseibacillus paracasei PC-01-co-fermented milk has more γ-aminobutyric acid and short-chain fatty acids than Lacticaseibacillus paracasei PC-01-fermented milk

Probiotics are increasingly used in the fermentation industry owing to their probiotic properties. However, most previous studies of milk fermentation are based on single-strain fermentation or focused on interactions between the starter strains without considering the role of probiotics in the microbial ecosystem. Thus, in this study, we compared the differences in the fermented milk metabolomes of single-strain fermentation with Lacticaseibacillus paracasei PC-01 (PC-01) versus dual-strain fermentation with both PC-01 and a probiotic, Bifidobacterium adolescentis B8589 (B8589), with the aim of revealing the metabolic effect of adding B8589 in the milk fermentation process. Although co-fermenting with B8589 did not influence the growth of PC-01, non-targeted metabolomics revealed significant changes in the dual-strain fermented milk metabolomes, characterized by increased levels of amino acid metabolites. Targeted metabolomics analysis confirmed that, by adding B8589 as the co-fermentation culture, the level of γ-aminobutyric acid increased by seven times; and the contents of acetate, propionate, butyrate, valerate, and citrate were also significantly enriched. Our results suggested that B8589 metabolically interacts with PC-01, which increases the amounts of functional metabolites in the co-fermented milk. This study provides interesting insights into the metabolic interaction between probiotic and starter culture in an industrial fermentation process.

Исследование влияния заквасок для производства йогурта на процесс ферментации козьего и коровьего молока в различных соотношениях

The purpose of the research is to study the effect of yogurt cultures on the efficiency of the goat milk fermentation process. The objects of research were goat's milk, cow's milk and starter cultures Bifidonorm from Lactoferm ECO (Italy) and bioprox YO-PROX 569 (France). The relevance of obtaining functional fermented dairy products based on goat's milk is substantiated. The optimal dosage of starter cultures in the production of yogurt based on goat's milk has been established. The effect of starter cultures on the fermentation process during fermentation of goat's and cow's milk in various ratios was studied. The possibility of using Bifidonorm and bioprox YO-PROX 569 starter cultures for the production of yogurt based on goat's milk has been confirmed. The relevance of functional fermented dairy products based on goat's milk obtaining is substantiated. The process of goat's and cow's milk mixtures fermentation was studied at their different ratio using two types of starter cultures. The influence of the direct vat set starter culture type on the organoleptic and physico-chemical parameters of yogurt based on goat's milk was studied. The possibility of goat’s milk using for the manufacturing of fermented dairy products has been confirmed.

Diversity analysis and gene function prediction of bacteria and fungi of Bactrian camel milk and naturally fermented camel milk from Alxa in Inner Mongolia

The microbial diversity in Bactrian camel milk and naturally fermented camel milk with unique nutritional properties and medicinal value in Alxa, Inner Mongolia, has been little reported. The results of this study showed that the dominant bacterial phyla in Bactrian camel milk and naturally fermented camel milk were Firmicutes and Proteobacteria, and dominant fungal phyla were Ascomycota and Basidiomycota. During the natural fermentation process of Bactrian camel milk, dominant bacterial genus changed from Lactococcus to Lactobacillus, while dominant fungal genera changed from Apiotrichum, Cutaneotrichosporon and Candida to Kazachstania and Kluyveromyces. Therefore, the diversity and flora structure of bacteria and fungi in Bactrian camel milk and naturally fermented camel milk were significantly different (P < 0.05). The predicted gene functions by PICRUSt2 software showed that the main metabolic genes of bacteria were global and overview maps, carbohydrate metabolism and amino acid metabolism, etc.; the main metabolic genes of fungi were glyoxylate cycle, aerobic respiration I and II, etc. And the relative abundance of bacteria related to food-borne pathogenicity and environmental pollution such as Enterobacter, Enterococcus and Raoultella, etc. suddenly decreased, which provides a reference for the safety evaluation of camel milk and camel milk products and a basis for the study of probiotics.

Expanding the genomes insight of Streptococcus thermophilus phages through a multifaceted approach

Viruses have complex evolutionary relationships, and several strategies have been used in an attempt to classify the phages that infect S. thermophilus. In this study, we used a wide range of complementary methods, including comparative genomics, core genome analysis, and signature genes phylogenetics, to show that the S. thermophilus phages are organized into 142 species and five genera (three of them new) and that due to their genetic diversity, the classification at family level varies according to the classification criteria used. No significantly conserved genes were identified among the 183 genomes evaluated. However, the genes encoding holin protein were conserved in more than 95% of genomes. The holins analysis suggests that at least two α-helix are required for protein function within S. thermophilus phages. This study expanded the of knowledge about the genetic diversity and evolution of streptococcal phages, both fundamental to promoting control strategies and minimizing failures in milk fermentation processes.

Rheological, microbiological and sensory properties of fermented khainak milk fermented with different starter cultures

A comparison study of the fermentation process of khainak (a hybrid of yak and cattle) and cow milk with commercial yoghurt (thermophilic), quark (mesophilic and mixed) starter cultures was conducted to find suitable starters using modern rheological, microbiological, and sensory methods. Lactic acid accumulation dynamic during the fermentation process of khainak milk with yoghurt starters was faster but did not differ from cow milk. The gel strength of the fermented khainak milk (206.3–447.2 Pa) was higher than that of cow milk (40–158 Pa). The total viable bacterial counts of all samples were not significantly different between the samples (8.43–9.46 log cfu mL−1). The improved whey separation in fermented khainak milk samples with mesophilic and mixed starter cultures suggests these starter cultures for the production of quark and cheese from khainak milk.

Pengaruh penambahan kacang hijau (vigna radiata (l.) R. Wilczek) pada fermensi susu oleh lactococcus lactis nbrc 12007

The high content of α-galactooligosaccharide in mung beans has the potential for supporting the growth of Lactic Acid Bacteria (LAB), therefore it can be developed as fermented plant-based milk that substituted Ultra High Temperature (UHT) milk. The purposes of this research is to investigate the effectiveness of LAB Lactococcus lactis NBRC 12007 in the fermentation process of mung bean milk and to observe the quality differences of fermented mung bean milk with UHT milk. Previously, preparations were made for starter L. lactis NBRC 12007 in MRSB medium and then sub cultured to UHT milk. Furthermore, 3% of the culture was inoculated into each of the mung bean milk substrate and UHT milk as a starter for the fermentation process and incubated for 24 hours at 37oC. During this process, the effectiveness of LAB was tested (total cell, total acid, total protein, pH, antibacterial) and the quality test of fermented products (organoleptic and physical). Based on total cells, total acid, total protein, pH, and antibacterial test findings, fermented mung bean milk products had a higher value than UHT milk products. During the process, it was proven that L. lactis NBRC 12007 had high effectiveness in producing and improving the quality of fermented mung bean milk compared to UHT milk. It is assumed that the content of oligosaccharides supports the growth of LAB and the fermentation process.

Dairy streptococcal cell wall and exopolysaccharide genome diversity.

The large-scale and high-intensity application of Streptococcus thermophilus species in milk fermentation processes is associated with a persistent threat of (bacterio)phage infection. Phage infection of starter cultures may cause inconsistent, slow or even failed fermentations with consequent diminished product quality and/or output. The phage life cycle commences with the recognition of, and binding to, a specific host-encoded and surface-exposed receptor, which in the case of S. thermophilus can be the rhamnose-glucose polysaccharide (RGP; specified by the rgp gene cluster) or exopolysaccharide (EPS; specified by the eps gene cluster). The genomic diversity of 23 S . thermophilus strains isolated from unpasteurized dairy products was evaluated, including a detailed analysis of the rgp and eps loci. In the present study, five novel eps genotypes were identified while variations of currently recognized rgp gene cluster types were also observed. Furthermore, the diversity of rgp genotypes amongst retrieved isolates positively correlated with phage diversity based on phageome analysis of eight representative dairy products. Our findings therefore substantially expand our knowledge on S. thermophilus’ strain and phage diversity in (artisanal) dairy products and highlight the merit of phageome analysis of artisanal and traditional fermented foods as a sensitive marker of dominant microbiota involved in the fermentation.

The application of extremely low-frequency (ELF) magnetic fields to accelerate the growth of Lactobacillus acidophilus L. bacteria and the milk fermentation process.

The long fermentation process makes the nutritional content of milk decrease due to spoilage. One of the causes of spoilage is the growth of pathogenic bacteria in milk. In regard to this, Lactobacillus acidophilus bacteria have proven effective as an antibacterial pathogen. This study aims to determine the magnetic flux density (MFD) and exposure time that result in the optimum growth of Lactobacillus acidophilus bacteria and their effect on fermented milk&rsquo;s lactic acid content and pH. The research sample was Lactobacillus acidophilus bacteria grown in cow&rsquo;s milk. The exposure given used a time-changing magnetic field (MF) with a frequency of 50 Hz and a MFD of 0.1 mT, 0.2 mT, and 0.3 mT for 0&ndash;25 minutes, respectively. The results showed that the optimum bacterial growth occurred in samples exposed to a MFD of 0.2 mT for 5 minutes. Meanwhile, inhibition of bacterial growth began on the exposure to a MFD of 0.3 mT for 15 minutes. Moreover, it was found that the highest lactic acid content and the lowest pH were obtained in exposed milk with a MFD of 0.2 mT for 5 minutes. Lactic acid content and pH were closely related to the number of Lactobacillus acidophilus bacteria colonies present in the milk.

The application of extremely low-frequency (ELF) magnetic fields to accelerate the growth of Lactobacillus acidophilus L. bacteria and the milk fermentation process [pdf

The application of extremely low-frequency (ELF) magnetic fields to accelerate the growth of Lactobacillus acidophilus L. bacteria and the milk fermentation process [pdf

Assessment of milk fermentation process at three temperatures using laser speckle technique

This research work contains information on speckle activity at different stages of the milk fermentation process at temperatures of 24 °C, 32 °C and 40 °C, using the dynamic laser speckle technique for the first time, to the best of our knowledge. Image processing algorithms such as inertia moment (IM), the absolute value of differences, the probability mass function of regular difference, spatial-temporal speckle correlation and 3D graphs of spectral images have been applied to show the speckle activity of the samples. All the methods show that the speckle activity of the sample is higher at the semisolid stage than at the liquid stage of the sample. Also, higher speckle activity of the curd has been observed at 40 °C (11 576.18 using IM) than at 32 °C (8550.92 using IM), followed by that at 24 °C (4855.88 using IM). At 40 °C, the thermal motion of curd particles is higher than that of the curd particles at the other two temperatures, and are responsible for the higher speckle activity. The preparation time for curd at 40 °C (5 h) is lower in comparison to the curds at 24 °C (10 h) and 32 °C (6 h). The lowest value for the titratable acidity of the curd prepared at 24 °C shows a lower % lactic acid than the curd at 32 °C and that at 40 °C. The lowest value of susceptibility to syneresis of the curd at 40 °C (30.00) represents a more compact curd than the curds at 32 °C (37.50) and 24 °C (45.00). The horizontal speckle grain size of the curd has been calculated using an autocovariance function; the highest is for curd fermented at 40 °C (12.34 μm), followed by that at 32 °C (11.59 μm), then that at 24 °C (10.96 μm).

Determination and standardization of the kefiran extraction protocol for possible pharmacological applications

The use of biopolymers in medicine and pharmacology is of great scientific, technological, and commercial interest. A possibility of biopolymer for use in pharmacological applications is the kefiran, an exopolysaccharide (EPS) produced by bacteria that form kefir grains during the milk fermentation process in which the grains are growing. The EPS extraction method can modify its concentration and structure. To establish the most appropriate kefiran extraction protocol and standardize this method, three different extraction processes were evaluated, which differ in the boiling times and in the presence (or not) of agitation. Kefiran obtained by different methods were characterized by thermogravimetric analysis (TGA), Fourier transform infrared spectroscopy (FTIR), electron scanning field emission microscopy (FESEM), and determination of antimicrobial potential. The samples showed differences in yield, chemical composition, degree of purity, thermal properties, and antimicrobial capacity. The results analyzed indicated that extraction protocol 1 (EP 1) with a longer heating time and constant agitation was able to provide the best thermal resistance, with a high degree of purity (93%), and yield (22.18 mg of kefiran obtained for each gram of kefir grains used). In addition, this method provides good biological activity as a potential antimicrobial agent and can be a potential biopolymer for pharmacological application.

Quality Analysis of Yoghurt from Goat's Milk Using Starter Lactic Acid Bacteria

Yoghurt is a pro-biotic beverage produced from the fermentation process of milk, namely from vegetable milk (soy milk) or animal milk (goat's milk and cow's milk). During the fermentation process, the chemical reactions that occur will turn milk into yogurt with the help of lactic acid bacteria. In the health sector, yogurt plays a role in increasing the body's immunity, digestive tract health and can prevent osteoporosis. In general, yogurt circulating in the community still has low nutritional content, so a more in-depth study needs to be done. The goal of this research is to examine the quality of yogurt by manipulating certain variables and adding other components to increase the yogurt's quality. Streptococcus thermophilus and Lactobacillus bulgaricus were used as starter as much as 12.5 mL each (10% of 500 mL of cream milk). The fixed variables in this study were the volume of goat's milk 500 ml, cream concentration 6%, pasteurization temperature 85°C, pasteurization time 15 minutes and fermentation temperature 45°C, while the independent variables were varying the length of the fermentation process for 3, 4, 5, 6, 7, 8 and 9 hours. From this study, it was concluded that yogurt with optimum results was obtained at a fermentation time of 6 hours, the pH value was 3.8, lactic acid content was 1.305%, protein content was 5.54%, fat content was 4.98%, and moisture content was 84.10%.

Probiotic characterization of Lactobacillus strains isolated from breast milk and employment for the elaboration of a fermented milk product

A total of seven strains of lactic acid bacteria (LAB) isolated from breast milk, were investigated for their probiotic properties. Lactobacillus plantarum 5H1 and 5L1 were selected for their beneficial properties and used for probiotic fermented milk manufacture. The fermentation parameters (pH and lactic acid) and LAB viability during fermented milk processing and storage were evaluated. The results showed that the strains had a broad spectrum of antimicrobial activity against pathogenic bacteria and toxigenic fungi. These also evidenced capacity of adhesion and reduction of Salmonella adhesion to Caco-2 cells. During milk fermentation process, an increase in lactic acid content, a decrease in milk pH, and an increase in the total bacterial count were evidenced. LAB viability in fermented milk during storage remained around 8 log10 CFU/mL. This workflow can be useful for the isolation of new probiotics and the development of fermented milks with probiotic properties to promote host health.

Screening of Streptococcus thermophilus with Good Flavor and Analysis of Its Aroma Production Characteristics

As one of the main strains of fermented milk, Streptococcus thermophilus can impart excellent texture, rich nutritional value and unique flavor to fermented milk during the fermentation and storage process of fermented milk. On the basis of the previous research, 7 strains of S. thermophilus with good fermentation characteristics isolated from traditional fermented milk in different regions were used as the experimental strains this experiment, and commercial starters were used as the control group, solid phase microextraction was used combined with gas chromatography-mass spectrometry technology to detect and analyze the volatile flavor substances in the fermented milk at the end of the fermentation, which was pH4.5. The results showed that among all the experimental strains, the composition and content of volatile flavor compounds in G80-2 fermented milk were closest to the control group. On this basis, the dynamic analysis of the volatile flavor substances produced by the G80-2 strain during the fermentation and storage of milk was carried out. It was found that the strain was detected acids (4 types), alcohols (10 types), ketones (12 types), aldehydes (6 types), esters (1 type) and other compounds during fermentation and storage. Some main characteristic flavor substances such as acetic acid, acetaldehyde, diacetyl, acetoin, 2-heptanone, 1-heptanol, etc. were relatively high in content. It was concluded that the strain could be used as a starter in the production of dairy products.

Dynamic Modeling of the Impact of Temperature Changes on CO2 Production during Milk Fermentation in Batch Bioreactors.

Knowledge of the mathematical models of the fermentation processes is indispensable for their simulation and optimization and for the design and synthesis of the applicable control systems. The paper focuses on determining a dynamic mathematical model of the milk fermentation process taking place in a batch bioreactor. Models in the literature describe milk fermentation in batch bioreactors as an autonomous system. They do not enable the analysis of the effect of temperature changes on the metabolism during fermentation. In the presented extensive multidisciplinary study, we have developed a new mathematical model that considers the impact of temperature changes on the dynamics of the CO2 produced during fermentation in the batch bioreactor. Based on laboratory tests and theoretical analysis, the appropriate structure of the temperature-considered dynamic model was first determined. Next, the model parameters of the fermentation process in the laboratory bioreactor were identified by means of particle swarm optimization. Finally, the experiments with the laboratory batch bioreactor were compared with the simulations to verify the derived mathematical model. The developed model proved to be very suitable for simulations, and, above all, it enables the design and synthesis of a control system for batch bioreactors.

Fortification of Acidophilus-bifidus-thermophilus (ABT) Fermented Milk with Heat-Treated Industrial Yeast Enhances Its Selected Properties.

The improvement of milk dairy products’ quality and nutritional value during shelf-life storage is the ultimate goal of many studies worldwide. Therefore, in the present study, prospective beneficial effects of adding two different industrial yeasts, Kluyveromyces lactis and Saccharomyces cerevisiae pretreated by heating at 85 °C for 10 min to be inactivated, before fermentation on some properties of ABT fermented milk were evaluated. The results of this study showed that the addition of 3% and 5% (w/v) heat-treated yeasts to the milk enhanced the growth of starter culture, Lactobacillus acidophilus, Bifidobacteria, and Streptococcus thermophilus, during the fermentation period as well as its viability after 20 days of cold storage at 5 ± 1 °C. Furthermore, levels of lactic and acetic acids were significantly increased from 120.45 ± 0.65 and 457.80 ± 0.70 µg/mL in the control without heat-treated yeast to 145.67 ± 0.77 and 488.32 ± 0.33 µg/mL with 5% supplementation of Sacch. cerevisiae respectively. Moreover, the addition of heat-treated yeasts to ABT fermented milk enhanced the antioxidant capacity by increasing the efficiency of free radical scavenging as well as the proteolytic activity. Taken together, these results suggest promising application of non-viable industrial yeasts as nutrients in the fermentation process of ABT milk to enhance the growth and viability of ABT starter cultures before and after a 20-day cold storage period by improving the fermented milk level of organic acids, antioxidant capacity, and proteolytic activities.