Natural Fermented Milk Research Articles

Comparative analysis of microbial succession and proteolysis focusing on amino acid pathways in Asiago-PDO cheese from two dairies

In this study, a comprehensive and comparative analysis was conducted on Italian Asiago-PDO cheese obtained from two different dairies named Dairy I and Dairy II using industrial and natural fermented milk, respectively. The analysis encompassed the evaluation of chemical composition, the succession of the microbiota during manufacture and ripening, and proteolysis mainly focusing on free individual amino acid (FAA) profiles. A metagenomic approach was used to investigate the cheese microbiome functionality. Differences in gross chemical composition were more evident during ripening, with Dairy II showing higher variability within batches. The microbiota varied significantly between the two dairies and ripening stages. The choice of starter culture shaped the microbiota during production and affected the microbial diversity of non-starter lactic acid bacteria (NSLAB) originated from the raw milk during ripening. Peptide chromatographic profiles and FAA concentrations increased as ripening progressed, with Dairy I showing higher production of FAA. Functional analysis of the metagenomes linked species to specific amino acid metabolism/catabolism pathways. The amino acid metabolism pathways, particularly those related to aromatic amino acids, lysine, and branched-chain amino acids, were affected by the presence of specific NSLAB species, which differed between the two dairies. The results obtained in this study reveal the impact of starter culture on peculiar cheese microbiota assemblies, which selectively targets amino acid pathways, providing insights into the potential flavor and aroma characteristics of Asiago-PDO cheese.

Genome Analysis of Potential Probiotic Levilactobacillus brevis AcCh91 Isolated from Indian Home-Made Fermented Milk Product (Chhurpi).

Consumption of naturally fermented milk (NFM) products is the dietary culture in India. The mountainous people of Arunachal Pradesh in India prepare the assorted artisanal home-made NFM products from cow and yak milk. Previously, we isolated and identified 76 strains of lactic acid bacteria (LAB) from NFM products of Arunachal Pradesh, viz. mar, chhurpi and churkam. We hypothesized that some of these LAB strains may possess probiotic potentials; hence, we investigated the probiotic potentials of these strains. On the basis of in vitro and genetic screening for probiotic attributes including haemolytic ability, 20 LAB strains were selected out of 76 strains, for further analysis. Using in silico analysis, viz. multivariate heatmap and PCA (principal component analysis) biplot, Levilactobacillus brevis AcCh91 was selected as the most promising probiotic strain, which was further characterized by the whole-genome analysis. Lev. brevis AcCh91 showed the highest survival rate of 93.38% in low pH and 86.68 ± 2.69% in low bile and the highest hydrophobicity average of 86.34 ± 5.53%. This strain also showed auto-aggregation and co-aggregation with antimicrobial properties against the pathogens, showed ability to produce beta-galactosidase and cholesterol reduction property and, most importantly, produced GABA, an important psychobiotic element. Genomic analysis of Lev. brevis AcCh91 showed the presence of genes corresponding to GABA, vitamins, amino acids, cholesterol reduction, immunomodulation, bioactive peptides and antioxidant activity. The absence of antimicrobial-resistant genes and virulence factors was observed. Hence, genome analysis supports the probiotic potentials of Lev. brevis AcCh91, which may be further investigated to understand its health-promoting properties.

Lactobacillus delbrueckii subsp. bulgaricus strain TCI904 reduces body weight gain, modulates immune response, improves metabolism and anxiety in high fat diet-induced obese mice

The multiple probiotic characteristics of strain TCI904 isolated in this study from natural fermented milk were investigated using a mouse model. TCI904 was identified as Lactobacillus delbrueckii subsp. bulgaricu (LDB), a well-known lactic acid starter bacterium found in yogurt. TCI904 exhibited an outstanding pancreatic lipase inhibition activity among several strains of lactic acid bacteria in vitro. Its in vivo effects were further studied. In a comparison of mice fed a high-fat diet (HFD) and those fed a HFD combined with TCI904 for 9 weeks, differences were observed in various aspects of health, and the adverse effects of a HFD were prevented in the latter group. TCI904 effectively prevented fat and body weight accumulation without reducing food intake; it also modulated innate immunity and increased the level of IgA in feces, reversing the increased blood sugar and insulin levels and attenuated the hyperlipidemia caused by a HFD. Based on biochemical test data, compared with the HFD group, a HFD combined with TCI904 induced significant lowering of insulin resistance indicator, homeostasis model assessment-insulin resistance (HOMA-IR) and atherogenic indices of plasma (AIP), the atherogenic coefficient (AC) and cardiac risk ratio (CRR) and increased the cardioprotective index (CPI). In addition, the administration of TCI904 alleviated mood disorders caused by a HFD. Taking the recommended human dose of TCI904 did not affect the liver or kidney function, indicating that TCI904 has sufficient in vivo safety. Taken together, the results of the present study contributed towards validation of the probiotic benefits of lactic acid starter microflora. Orally taken TCI904 exhibited positive immune- and metabolic-modulating, and anxiolytic properties, especially in HFD-induced obesity.

Phenotype testing, genome analysis, and metabolic interactions of three lactic acid bacteria strains existing as a consortium in a naturally fermented milk.

This work reports the characterization of three lactic acid bacteria (LAB) strains –Lactococcus lactis LA1, Lactococcus cremoris LA10, and Lactiplantibacillus plantarum LA30– existing as a stable consortium in a backslopping-inoculated, naturally fermented milk (NFM). This study aimed at uncovering the biochemical and genetic basis of the stability of the consortium and the cooperativity among the strains during milk fermentation. All three strains were subjected to phenotyping, covering the utilization of carbohydrates, enzyme activity, and antibiotic resistance. The strains were grown in milk individually, as well as in all possible combinations, and the resulting fermented product was analyzed for sugars, organic acids, and volatile compounds. Finally, the genomes of the three strains were sequenced and analyzed for genes associated with technological and safety properties. As expected, wide phenotypic diversity was seen between the strains. Lactococcus cremoris LA10 was the only strain to reach high cell densities and coagulate milk alone after incubation at 22°C for 24 h; congruently, it possessed a gene coding for a PrtP type II caseinolytic protease. Compared to any other fermentation, acetaldehyde concentrations were greater by a factor of six when all three strains grew together in milk, suggesting that its production might be the result of an interaction between them. Lactococcus lactis LA1, which carried a plasmid-encoded citQRP operon, was able to utilize milk citrate producing diacetyl and acetoin. No genes encoding virulence traits or pathogenicity factors were identified in any of the strains, and none produced biogenic amines from amino acid precursors, suggesting them to be safe. Lactiplantibacillus plantarum LA30 was susceptible to tetracycline, although it harbors a disrupted antibiotic resistance gene belonging to the tetM/tetW/tetO/tetS family. All three strains contained large numbers of pseudogenes, suggesting that they are well adapted (“domesticated”) to the milk environment. The consortium as a whole or its individual strains might have a use as a starter or as starter components for dairy fermentations. The study of simple consortia, such as that existing in this NFM, can help reveal how microorganisms interact with one another, and what influence they may have on the sensorial properties of fermented products.

Rapid isolation of exopolysaccharide-producing Streptococcus thermophilus based on molecular marker screening.

As a natural food additive, exopolysaccharide (EPS) produced by Streptococcus thermophilus can improve product viscosity and texture. The protein EpsA is a putative pathway-specific transcriptional regulator for EPS biosynthesis in S.thermophilus. According to comparative analysis of EPS biosynthetic gene clusters, a conserved region of epsA (609 bp) was employed to design primer pair epsA-F/R as a molecular marker for the isolation of EPS-producing (EPS+ ) S. thermophilus. Two EPS+ S. thermophiles strains, AR333 and S-3, were band-positive, whereas Lactococcus lactis NZ9000 (non-EPS-producing, EPS- ), Lactobacillus casei LC2W (EPS+ ) and L. plantarum AR113 (EPS+ ) were negative by polymerase chain reaction (PCR) amplicon bands using the epsA probe. This indicated good specificity of the epsA probe to EPS+ S. thermophilus. Moreover, based on PCR screening with the epsA probe, 23 positive strains were isolated and identified as S. thermophilus from our microbial library and natural fermented milk with 141.3-309.2mg L-1 of EPS production, demonstrating the validity of our molecular marker screening method. The designed molecular marker of epsA can rapidly screen EPS+ S. thermophilus, which has potential application in the dairy and other food industries. © 2021 Society of Chemical Industry.

Detection of sorbate potassium in Brazilian commercial fermented milks

Este estudo teve o objetivo de determinar a presença de ácido sórbico e seus sais em leites fermentados comercializados no Brasil, bem como quantificá-lo quando a amostra foi positiva. Vinte e seis amostras comerciais foram analisadas (n = 3), totalizando 78 amostras. As amostras foram divididas em dois grupos: amostras naturais (5) e com adição de ingredientes não lácteos (21). Um total de 61,53% amostras foram positivas para ácido sórbico e seus sais. Nestes, 3,85% eram de leites fermentados naturais, o que segundo o Codex Alimentarius e a legislação brasileira é caracterizado como fraude. Além disso, todas as amostras estavam fora da concentração máxima de sorbato de potássio de acordo com a legislação brasileira (0,3 g.kg-1) vigente, no entanto, estavam dentro dos parâmetros aceitáveis pelo Codex Alimentarius (1,0 g.kg-1). Podemos concluir que o ácido sórbico e seus sais foram detectados em leite fermentado natural e com ingredientes não lácteos.

Bio-Removal of Methylene Blue from Aqueous Solution by Galactomyces geotrichum KL20A

The conventional treatments used to remove dyes produced as a result of different industrial activities are not completely effective. At times, some toxic by-products are generated, affecting aquatic ecosystems. In this article, an efficient use of microorganisms is presented as a biodegradation technique that is a safe environmental alternative for the benefit of aquatic life. A strain of the yeast Galactomyces geotrichum KL20A isolated from Kumis (a Colombian natural fermented milk) was used for Methylene Blue (MB) bioremoval. Two parameters of the bioremediation process were studied at three different levels: initial dye concentration and growth temperature. The maximum time of MB exposure to the yeast was 48 h. Finally, a pseudo-first-order model was used to simulate the kinetics of the process. The removal percentages of MB, by action of G. geotrichum KL20A were greater than 70% under the best operating conditions and in addition, the kinetic simulation of the experimental results indicated that the constant rate of the process was 2.2 × 10-2 h−1 with a half time for biotransformation of 31.2 h. The cytotoxicity test based on the hemolytic reaction indicated that by-products obtained after the bioremoval process reached a much lower percentage of hemolysis (22%) compared to the hemolytic activity of the negative control (100%). All of these results suggest that the strain has the capacity to remove significant amounts of MB from wastewater effluents.

Bacterial community in naturally fermented milk products of Arunachal Pradesh and Sikkim of India analysed by high-throughput amplicon sequencing

Naturally fermented milk (NFM) products are popular ethnic fermented foods in Arunachal Pradesh and Sikkim states of India. The present study is the first to have documented the bacterial community in 54 samples of NFM products viz. chhurpi, churkam, dahi and gheu/mar by high-throughput Illumina amplicon sequencing. Metagenomic investigation showed that Firmicutes (Streptococcaceae, Lactobacillaceae) and Proteobacteria (Acetobacteraceae) were the two predominant members of the bacterial communities in these products. Lactococcus lactis and Lactobacillus helveticus were the predominant lactic acid bacteria while Acetobacter spp. and Gluconobacter spp. were the predominant acetic acid bacteria present in these products.

Some Technological Properties of Lactic Acid Bacteria Isolated from Dahi and Datshi, Naturally Fermented Milk Products of Bhutan.

Dahi and datshi are common naturally fermented milk (NFM) products of Bhutan. Population of lactic acid bacteria (LAB) in dahi (pH 3.7) and datshi (pH 5.2) was 1.4 × 107 and 3.9 × 108 cfu/ml, respectively. Based on 16S rRNA gene sequencing isolates of LAB from dahi and datshi were identified as Enterococcus faecalis, E. faecium, Lactococcus lactis subsp. lactis. LAB strains were tested for some technological properties. All LAB strains except E. faecalis CH2:17 caused coagulation of milk at both 30°C for 48 h. Only E. faecium DH4:05 strain was resistant to pH 3. No significant difference (P > 0.05) of viable counts was observed in MRS broth with and without lysozyme. All LAB strains grew well in 0.3% bile showing their ability to tolerate bile salt. None of the LAB strains showed >70% hydrophobicity. This study, being the first of its microbiological analysis of the NFM of Bhutan, has opened up to an extent of research work that gives a new insight to the products.

Naturally fermented milk products of the Eastern Himalayas

BackgroundPastoralists comprising different ethnic groups of people dominate the Eastern Himalayas. Traditional knowledge in the Eastern Himalayas reflects the common linkage of origin and settlement of the ethnic groups in the regions. The practice of milk fermentation along the Eastern Himalayan regions shows similar types of ethnic naturally fermented milk (NFM) products that are regularly prepared by different ethnic groups of people. MethodsA survey of various types of NFM products of Eastern Nepal, Darjeeling Hills, Sikkim, and Arunachal Pradesh in India, and Bhutan and their methods of preparation, mode of consumption, and ethnic values was documented as per the standard method. ResultsSikkim and Nepal have several varieties of NFM products, which include dahi, mohi, gheu, soft chhurpi, hard chhurpi, dudh-chhurpi, chhu, somar, maa, philu, and shyow. The main products, which are daily prepared in Arunachal Pradesh, are mar, chhurpi/churapi, churkam, and churtang/chhurpupu. NFM products of Bhutan are dahi, datshi, mohi, gheu, chugo, and hitpa. ConclusionUnique types of NFM products have been reported from the Eastern Himalayas. Although these are minor products, they are of high biological importance.

イエメン産の伝統的自然発酵乳“ラバン”の菌叢ならびに官能特性の解析

イエメン産の伝統的自然発酵乳“ラバン”の菌叢ならびに官能特性の解析

Mixes of Carrot Juice and Some Fermented Dairy Products: Potentiality as Novel Functional Beverages

The present study aimed to formulate and evaluate yoghurt and Rayeb (Traditional Egyptian natural fermented milk) mixes with red and yellow carrot juices. Out of 16 different mixing ratios (Eight for yoghurt and eight for Rayeb), data indicated that the most significantly acceptable mixes as judged by panelists were: yoghurt-red carrot juice (3:1 v/v), yoghurt-yellow carrot juice (2:1 v/v), Rayeb-red carrot juice (2:1 v/v) and Rayeb-yellow carrot juice (4:1 v/v). The aforementioned mixes were further investigated versus their counterpart controls (yoghurt and Rayeb). It was obvious that the formulated mixes contained considerably higher concentrations of the bioactive components mainly, ascorbic acid and anthocyanins (red-carrot mixes) and β-carotene (yellow-carrot mixes) along with antioxidant activity (DMPD Radical Scavenging Activity) and higher counts of probiotic LAB, as compared to the controls.

Development of of prebiotic fermented goat milk, with low lactose added β-cyclodextrin

Consumers are interested, actually, in foods besides nutritive healthy. Goat milk and its derivatives play an important role as a source of calcium, high digestibility fat, high biological value and hypoallergenicity protein. Dealing the demand for healthy dairy derivatives, a small dairy in Coronel Pacheco has made fermented milk natural, facing, however, serious difficulties in the coagulation of milk from his herd. The product formed a very fine and fluid curd. Another difficulty faced was the characteristic goat flavor of milk which reduced the acceptability of fermented milk among its consumers. Thus, the objective of this study was to optimize the manufacturing process of natural fermented milk for this dairy, seeking to solve the difficulty of this derived milk clotting. Different treatments were tested in order to make the curd formed by coagulation of milk more consistent and improve fermented milk sensory acceptability. From preliminary tests and physical-chemical and microbiological analysis of raw material, two formulations were developed of concentrate fermented milk, one added only of inulin and other added of inulin, β-cyclodextrinand lactase. Was also performed a physicalchemical and microbiological analysis of the developed formulations and evaluated the sensory acceptability of these formulations for flavor, aroma, texture and overall acceptance using a hedonic scale of nine points. The post acidification and the viable lactic acid bacteria count of the formulations were followed for 30 days. It was observed that the process of concentration by evaporation and addition of inulin was efficient to make the produced curd more consistent. It was also found that the two formulations of fermented milk were according to the legal standards in relation to physico-chemical and microbiological requirements of the Normative Instruction no 46 of 2007, which establishes the Technical Regulation of Identity and Quality of Fermented Milks.They also maintained their sensory characteristics related to flavor and aroma, and regard the acidity and lactic acid bacteria counts requirements of the Standard of Identity and Quality of Fermented Milk during 30 days of storage at 7oC. The formulation of fermented milk concentrate with insulin, β-cyclodextrin and lactase showed greater acceptance for flavor and overall impression than the formulation of fermented milk concentrate with only inulin (p< 0.01). In relation to the texture and aroma, the two formulations presented the same acceptance (p< 0.01). The average acceptance of the formulation of fermented milk concentrate with added inulin, β-cyclodextrin and lactase in flavor was situated between the hedonic terms indifferent and like slightly and in relation to attributes: flavor, texture and overall impression were ranked among the hedonic terms like slightly and like moderately, indicating that the product had good acceptance.

Technological characterization of lactococci isolated from traditional Chinese fermented milks

To screen lactic acid bacteria for starter cultures in cheese production, 21 Lactococcus strains previously isolated from natural fermented milk and koumiss made by herdsman families in the Xinjiang, Gansu, and Qinghai provinces of China were evaluated for optimal growth temperature, acidification activity, proteolytic activity, aminopeptidase activity, and autolytic activity. All isolates presented low acidification rates, and the pH value did not reach 5.3 after 6h of inoculation in sterile reconstituted skim milk at 30°C. Strains X9C2 and T7C showed the highest proteolytic activity of 24.67 and 23.58mg of glycine/L of milk, respectively. For aminopeptidase activity, strains X9C2 and T1C2 displayed the highest activities of 30.56 and 27.70 U/mg of protein using l-leucine-p-nitroanilide as substrate, respectively. Autolytic activity in simulated cheese-like buffer ranged from 7.45 to 34.76%, and strains Q14C2 and Q16C showed the highest values of 34.76 and 34.20%, respectively. Collectively, one main finding is that some technological characteristics of Lactococcus isolates from Chinese traditional fermented products varied greatly. Some isolates with potentially important properties could be valuable for application as starter cultures of cheese or could constitute a mixed culture.

Microbial characterisation and stability of a farmhouse natural fermented milk from Spain

This work reports the microbial characterisation of a farmhouse natural fermented milk (NFM) with good sensorial properties produced in Spain. Culturing and denaturing gradient gel electrophoresis (DGGE) analyses showed thatLactococcus lactissubsp.lactis and L. lactissubsp.cremoris(approximate levels of 109 cfu/mL) were dominant in this NFM, whileLactobacillus plantarumappeared at a lower level (106–107 cfu/mL). Repetitive extragenic palindromic (REP)‐PCR typing of the isolates identified single strains each ofLc. lactissubsp.lactis, Lc. lactissubsp.cremorisandLb. plantarum. These three strains formed a stable microbial association which has been maintained for at least some decades.

The effect of natural fermented milk in the protection of liver from cancer

PurposeThe purpose of this paper is to evaluate the protective effect of natural fermented milk (NFM) against liver cancer.Design/methodology/approachFive types of NFM were collected from different places around Makka. The natural flora which present in them were studied and certain types which contain Lactobacillus spp. and Bifidobacterium spp. strains were selected to be used in the research. The nutritional experiment was carried out on 20 Albino mice divided into four groups. The first group (A) was control (not fed on NFM). The second (B) and third (C) groups were fed daily on (NFM) for two weeks. The final group (D) received basal diet only for four weeks, but after two weeks the third and fourth groups were treated orally by one dose of toxic mutagen (0.25 mg/mouse). The quality of life was measured by: specific strain count in stool; histopathological and histochemical studies on liver; and lymphocytic count.FindingsCow's milk which contains Lactobacillus spp. and Bifidobacterium spp. has a protective effect depending on its anti‐microbial properties. Also it enhances the endogenous intestinal probiotic bacteria that have several beneficial effects (i.e. detoxification and antigen toxicity) and stimulation of immune modulator cells.Originality/valueThe paper evaluates the protective effect of natural fermented milk (NFM) against cancer of the liver.

Antimicrobial properties of lactic acid bacteria and yeast-LAB cultures isolated from traditional fermented milk against pathogenic Escherichia coli and Salmonella enteritidis strains

The survival and growth of Escherichia coli 3339 and Salmonella enteritidis 949575 isolated from human clinical samples, in milk fermented with lactic acid bacteria (LAB) and yeast strains previously isolated from Zimbabwean naturally fermented milk (NFM) was studied. The LAB starter cultures used were Lactococcus lactis subsp. lactis biovar. diacetylactis C1 alone (C1) or in combination with Candida kefyr 23 (C1/23), L. lactis subsp. lactis Lc261 alone (LC261) or in combination with C. kefyr 23 (Lc261/23). The growth of the same pathogens in milk fermented with a commercial DL culture (CH-N 22) and spontaneously fermented raw milk was also monitored. The C1 and C1/23 cultures significantly ( P < 0.05) inhibited the growth of both pathogens. When inoculated at the beginning of the fermentation, both E. coli 3339 and S. enteritidis 949575 counts were significantly ( P < 0.05) reduced by about two log cycles in C1 and C1/23 cultured milk. However, in naturally fermented milk and the DL cultured milk, both E. coli 3339 and S. enteritidis 949575 grew and reached high populations of about 9 and 8.8 log cfu ml − 1 , respectively, after 18 h. When E. coli 3339 was inoculated into previously fermented milk, the viable counts were significantly ( P < 0.05) reduced in the presence of C1 and C1/23 from 7 log cfu ml − 1 to 3 log cfu ml − 1 after 48 h. S. enteritidis 949575 could not be recovered from these cultures after 48 h. The addition of the yeast did not enhance or diminish the inhibitory capacity of the LAB cultures. The pathogens survived in high numbers when inoculated into pre-fermented NFM and the commercial DL- (CH-N 22) cultured milk. The C1 strain, therefore, offered the best protection against the pathogens. Its inhibitory effect was mainly related to fast acid production.