Diversity of Lactic Acid Bacteria in Senduro Goat Milk

The diversity of lactic acid bacteria (LAB) species in donkey's milk was analysed by culture-dependent microbial techniques. Dominant strains were isolated on agar media generally used for enumerating LAB. To enrich the number of acidifying LAB present, the milk samples were incubated at 37°C for 24h (cultured milk samples, CM). One of the CM samples was heat-treated at 63°C for 10min before incubation at 37°C (heat-treated and cultured milk sample, TCM) to select thermophilic LAB. The microflora in these CM and TCM samples was then compared to that of the raw milk samples (RM). Among the 129 LAB isolates, 10 different species (four Enterococcus, five Streptococcus and one Pediococcus) were identified by molecular methods. Although the 10 LAB species were present in the RM samples, only three and two isolates were found in CM and TCM samples, respectively. Despite the selection protocol being set up to favour the isolation of all LAB isolates present in donkey milk, relatively few species and biotypes were isolated. No LAB isolates belonging to the most technologically important dairy starter species were detected. The possible factors related to the limited LAB diversity in donkey's milk have been discussed below. There is increased interest in using donkey's milk as a source of human nutrition. The large amounts of antimicrobial components and defence factors present in donkey's milk provide protection from microbial infections and distinguish donkey's milk from the milks of other mammals. However, the microbiota in donkey's milk has so far been poorly characterized, specifically with regard to the lactic acid bacteria (LAB). This study has identified cultivable, acidifying and thermoduric LAB that could be used to develop starter cultures. This is the first study to investigate the culturable LAB microbiota present in donkey's milk.

Distribution and genetic diversity of lactic acid bacteria from traditional fermented sausage

Defined as an artisanal curdled milk, Nonnonkoumou is a commodity appreciated by the Ivorian population, particularly that of the city of Daloa. In this food, there are a diversity of lactic acid bacteria (LAB) which play a very important role in fermented products. The objective of this work was therefore to identify the LAB isolated during the Nonnonkoumou production in order to get to know them better and use them for biotechnological purposes. LAB were isolated from fermenting raw milk used for Nonnonkoumou production at different fermentation times (T0, T6H, T12H, T18H and T24H). These LAB were characterized after Rep-PCR followed by partial sequencing of the 16S rRNA gene. The loads of LAB from Nonnonkoumou samples increase of 1.27 ± 0.12 log CFU / mL at 7.11 ± 0.43 log CFU / mL between 0 and 12 hours of fermentation. The maximum value (8.56 ± 0.18 log CFU / mL) is reached at 18 h of fermentation. The results of the Rep-PCR profile of 179 isolates revealed 8 distinct groups of LAB in Nonnonkoumou. After amplification and partial sequencing of the 16S rRNA gene of LAB strains, the 179 LAB isolates were classified into 8 species: Lactococcus lactis subsp cremoris (0.56%), Lactococcus Taiwanensis (16.20%), Weissella fabalis (12.29%), Wessiella confuse (3.35%), Lactiplantibacillus (10.06%), Weissella Hellenica (18.43%), Weissella paramesenteroides (1.68%) and Levilactobacillus brevis (37.43%). Thus, the study of this dynamics has made it possible to observe the natural variations in the composition of the LAB communities.

Purpose: Goat’s milk is enriched with many nutrients and diverse lactic acid bacteria (LAB). This research was aimed at isolating and characterizing industrially beneficial probiotic lactic acid bacteria present in goat milk and to investigate their ability to produce lactic acid.Research Method: A total of 100 bacterial isolates were obtained from 20 fresh goat’s milk samples from Western and North Western provinces in Sri Lanka. These isolates were characterized based on their colony and cell morphologies, physiological and biochemical characteristics, tolerance to in-vitro gastro intestinal tract (GIT) conditions and industrial properties. Species level identification of the isolates was performed based on the obtained phenotypic properties according to the Bergey’s manual of systematic bacteriology.Findings: The selected eight probiotic LAB isolates did not produce hemolytic enzymes, therefore, could primarily consider as safe for live consumption. According to the phenotypic properties, the isolates were of Lactobacillus pentosus, Lactobacillus plantarum, Streptococcus thermophilus, Streptococcus bovis, Lactococcus lactis and Enterococcus faecium species. They were able to coagulate skimmed milk which was indicated by the decrease of fi nal pH (4.8-5.9), tolerate to high (45 °C) and low (15°C) temperatures, grow in different NaCl concentrations (2%, 4%, and 6.5%) and different pH levels (1.5-3.9). HPLC analysis revealed that, Lactobacillus pentosus (33 mg/L) and Streptococcus thermophiles (14mg/L) possessed the highest lactic acid production abilities.Research Limitations: The species level identification of the isolates should be continued employing genotypic methods. The safety of the isolates for live consumption should confirm with in-vivo methods.Originality/Value: The raw goat milk microbiota is considered as a good source of novel lactic acid bacteria (LAB) strains that can be exploited for use as industrial starters and probiotics. Lactic acid produced by the microorganisms; specifically, lactic acid bacteria is an important commodity chemical having many numbers of applications. However, very less research work has been done on the characterization of beneficial lactic acid bacteria present in goat milk produced in Sri Lanka.

Diversity of non-starter lactic acid bacteria in autochthonous dairy products from Western Balkan Countries - Technological and probiotic properties

Aim. To isolate and identify bacteria with antagonist properties for biocontrol of the agents of bacterial diseases of soryz (Sorghum oryzoidum) and sorghum crops. Methods. The studies were conducted in 2021-2023. Spore-forming bacteria were isolated from the soryz samples, collected in the fields of the experimental farm of the Uman National Horticulture University (Cherkasy region, Uman). Lactic acid bacteria were isolated from soryz plants, collected in the private land plot, located between the villages of Teolyn, Vladyslavchyk, Kniazhyky in Monastyryshche com- munity, Uman district, where Pershotravneve hamlet used to be situated. A total of 1,250 samples were analyzed. The experiment had three repeats. Spore-forming and lactic acid bacteria were isolated from the surface of soryz plants on the firm ripe stage in summer while isolating phytopathogenic bacteria. The isolates of lactic acid bacteria- antagonists were also isolated from the inner part of winter stubble stalk of soryz, collected from the tilled field. The antagonistic activity of the strains of lactic acid bacteria and spore-forming bacteria, isolated from different ecological niches, to phytopathogens of soryz and sorghum crops was determined in vitro. The strains of Pseudomonas syringae, the agents of soryz bacterial spots, were used as test-cultures: 211141a, 211141, 210341, 21034, and 210521, along with the collection strains of phytopathogens: Pseudomonas syringae 8299, Pseudomonas syringae subsp. syringae UKM B-1021, X. oryzae 8375, Dickeya chrysanthemi 8683, Diskeya chryzanthemi 8683. The antagonistic activity of the extracted isolates of spore-forming and lactic acid bacteria was studied using the method of radial strokes (joint cultivation of the antagonist and the strains under investigation). The bacterial isolates were deemed inactive if the growth delay zone was 0–5 mm (–), from 5 to 10 mm (+) – low activity, 11–20 mm (++) – moderate activity, over 20 mm (+++) – high activity regarding the test-cultures. To check the effect of the isolate-antagonist of phytopathogenic bacteria, artificial infecting was conducted in the field conditions. For this purpose, a diurnal culture of the antagonist was introduced into the stalk of plants in the concentration of 1×108 colony-forming units, and 24 h later, a culture of test-strain of the phytopathogen was administered above the previous puncture. The results were evaluated 7–14 days after the artificial infection. The experiment had three repeats. The isolates of bacteria which demonstrated their an- tagonistic properties regarding the phytopathogenic bacteria were identified by their morphological properties, Gram staining, catalase test, profile of carbohydrate fermentation and mass-spectrometry (MALDI-TOF – Matrix Assisted Laser Desorption/Ionization) using VITEK MS mass-spectrometer. Results. Thirty-eight spore-forming bacterial iso- lates were extracted from soryz; among these, 21030, 21095, 21040, ASV1, ASV3, B4 demonstrated their antagonistic activity towards the investigated phytopathogenic bacteria. Isolate 21040 showed high antagonistic activity to most test-strains of P. syringae from soryz (the zone of negative culture – 23–30 mm) and lower activity regarding the collection cultures. Isolates B4 and AVS3 demonstrated their selective activity regarding the investigated phytopatho- gens. Twenty isolates of lactic acid bacteria were extracted. Higher antagonistic activity was noted for the isolates of lactic acid bacteria 8/1 and F1 to the strains of P. syringae, isolated from soryz and collection cultures. The highest antagonistic activity of isolate 8/1 was noted regarding test-strains of P. syringae 210521 and X. oryzae 8375 (the zone of negative culture – 40–35 mm). In the field conditions, the treatment of sorghum plants with F1 affected the pathological process that developed due to the impact of the phytopathogenic bacteria P. syringae, which led to the reduction in disease symptoms. The taxonomic position of the isolates of bacteria, which seem to be promising for the control of disease agents, was determined. In terms of morphology of cells and colonies, the biochemical profile, and mass-spectrometry MALDI-TOF, the spore-forming isolates 21040 and B4 were identified as Bacillus subtilis, and ASV3 – as Bacillus vallismortis. The identified isolates of lactic acid bacteria were Lactobacillus pentosus F1 and Lactobacillus sakei 8/1. Conclusions. In addition to phytopathogenic bacteria, from soryz plants we isolated the strains of spore-forming bacteria Bacillus subtilis 21040, B4, Bacillus vallismortis AVS3 and such lactic acid bacteria as Lactiplantibacillus pentosus and Lactobacillus sakei 8/1 (Latilactobacillus sakei 8/1), promising for the elaboration of methods for the biocontrol of the agents of bacterial diseases.

The water buffalo mozzarella cheese is a typical Italian cheese which has been introduced in the thriving Brazilian market in the last 10 y, with good acceptance by its consumers. Lactic acid bacteria (LAB) play an important role in the technological and sensory quality of mozzarella cheese. In this study, the aim was to evaluate the diversity of the autochthones viable LAB isolated from water buffalo mozzarella cheese under storage. Samples were collected in 3 independent trials in a dairy industry located in the southeast region of Brazil, on the 28th day of storage, at 4 ºC. The LAB were characterized by Gram staining, catalase test, capacity to assimilate citrate, and production of CO2 from glucose. The diversity of LAB was evaluated by RAPD-PCR (randomly amplified polymorphic DNA-polymerase chain reaction), 16S rRNA gene sequencing, and by Vitek 2 system. Twenty LAB strains were isolated and clustered into 12 different clusters, and identified as Streptococcus thermophilus, Enterococcus faecium, Enterococcus durans, Leuconostoc mesenteroides subsp. mesenteroides, Lactobacillus fermentum, Lactobacillus casei, Lactobacillus delbrueckii subsp. bulgaricus, and Lactobacillus helveticus. Enterococcus species were dominant and citrate-positive. Only the strains of L. mesenteroides subsp. mesenteroides and L. fermentum produced CO2 from glucose and were citrate-positive, while L. casei was only citrate positive. This is the first report which elucidates the LAB diversity involved in Brazilian water buffalo mozzarella cheese. Furthermore, the results show that despite the absence of natural whey cultures as starters in production, the LAB species identified are the ones typically found in mozzarella cheese.

Aims: Selective enumeration, isolation and characterization of lactic acid bacteria from different raw milk sources with special elucidation to lactic acid production. Study Design: Serial dilution for strain isolation from raw milk samples to be done followed by complete morphological, biochemical and molecular characterization aiming to study the microbial behavior to ferment different sugar and producing lactic acid as end product. Place and Duration of Study: Microbiology Biotechnology Laboratory, Tropilite Foods Pvt. Ltd. Gwalior (M.P)-INDIA from Sep 2012 to April 2013. Methodology: Milk samples from cow, sheep, goat, camel and buffalo were collected from the surrounding area of Gwalior district of Madhya Pradesh, India, from local milk suppliers. 13 potent strains in terms of lactic acid production were selected for analysis. Gram Research Article British Microbiology Research Journal, 3(4): 743-752, 2013 744 Staining, Catalase activity, Sugar fermentation, growth at high (45oC) and low (10oC) temperatures along with growth in different NaCl concentrations was observed with all isolates. Significant molecular characterization was done to determine the homology between different isolates of lactic acid bacteria. Results and Conclusion: Five potent lactic acid bacteria strains Streptococcus thermophilus from goat milk, Lactococcus lactis from buffalo milk, Streptococcus gallolyticus from camel milk, Streptococcus thermophilus from cow milk, Lactobacillus delbrueckii from sheep milk were identified capable of producing lactic acid in generous amount. Also all isolated strains from goat milk were found efficient in terms of lactic acid production when compared to other raw milk sources.

Microbiological and Food Safety Aspects of Tempeh Production in Indonesia

Bakasang is a fermented fish product traditionally made using the viscera (internal organs) of tuna (Thunnus spp.) and skipjack tuna (Katsuwonus pelamis), with the addition of a high concentration of salt, typically around 20% or more. The fermentation process occurs naturally and spontaneously through the activity of indigenous microorganisms, particularly those belonging to the lactic acid bacteria (LAB) group. LAB are Gram-positive, catalase-negative bacteria capable of producing lactic acid, which plays a crucial role in preservation and flavor development, and also possesses potential probiotic functionality. This study aims to evaluate the diversity and population of LAB in locally produced bakasang from the Bacan and Sanana regions, as a basis for the development of functional foods utilizing local microbial resources. Enumeration and isolation of LAB were conducted using the pour plate method on de Man, Rogosa, and Sharpe (MRS) agar, followed by morphological colony characterization, Gram staining, and physiological profiling. The results showed that LAB populations in bakasang samples from both regions ranged from 10⁵ to 10⁸ CFU/g. The LAB count in bakasang from Bacan (2.4 × 10⁸ CFU/g) was higher compared to that from Sanana (4.6 × 10⁵ CFU/g). Macroscopic, microscopic, and physiological characterization of the isolates indicated similarities to the genera Lactobacillus and Lactococcus. The diversity of LAB observed in this study reflects local microbiota with promising potential to be developed as fermentation agents and probiotics for the functional food industry.

The purpose of this study was to isolate and identify the lactic acid bacteria found in Sumba mares milk. This research will be carried out from September to November 2017 at the Veterinary Laboratory of the Livestock Service Office of East Nusa Tenggara Province. The research stages are isolation of lactic acid bacteria from Sumba mares milk by growing lactic acid bacteria on MRS agar media, and identifying lactic acid bacteria with gram staining, catalase test, motility test, and total plate count testing. Based on the results of the study, the presence of lactic acid bacteria originating from Sumba mares milk was characterized by the presence of colonies of lactic acid bacteria from Sumba mares milk which grew on MRS media agar as a selective medium for lactic acid bacteria. Based on the results of testing the characteristics of lactic acid bacteria Sumba mares milk has Gram positive, the negative results in the catalase test are characterized by not forming gas bubbles when the bacteria are added with H2O2. Based on the motility test obtained negative results or bacteria are non-motile and the number of lactic acid bacteria that can be a total picture of lactic acid bacteria that exist in Sumba mares milk in milliliter of milk. The conclusion are lactic acid bacteria isolated from Sumba mares milk has characteristic as Gram positive, basil or stem shaped, negative catalase and non motile, and has a total lactic acid bacteria of 3.5 x 108 cfu/ml (est).
 
 

This study aimed to characterize the diversity of lactic acid bacteria (LAB) isolated from fermented mare’s milk based on PCR-RFLP. The LAB was isolated from three fermented mare’s milk products from three different regions i.e Dompu, Bima, and Sumbawa, Province of West Nusa Tenggara, Indonesia. Fermented milk samples (25 mL) was diluted into 225 mL of saline solution and spread plated onto MRS and M17 agar containing 1 % CaCO3, then incubated at 37 ºC for 48 hours. Single colonies showing a clear zone were randomly selected and purified. The DNA was extracted by the heat-treatment method and amplified using an internal transcribed spacer (ITS) and 5.8S region spacer primers. The amplicons of the 5.8S region spacer were used for restriction fragment length polymorphism (RFLP) analysis using HaeIII and HindIII enzymes. The DNA bands were characterized to obtain the LAB clusters. The representative of each LAB cluster was amplified using 16S rDNA primer then sequenced. Total of 41 isolates was grouped into 10 clusters based on the PCR-RFLP analysis. However, only three clusters were identified as LAB from Lactobacillus genus. The Lactobacillus species obtained were L. plantarum and two strains of L. rhamnosus. Four clusters were identified as Staphylococcus genus and three others were Ochrobactrum genus. Thus, the LAB isolates from fermented mare milk obtained were the member of Lactobacillus genus. However, the Gram-negative bacteria and Staphylococcus were still found in the fermented products indicating the poor quality of the products. Therefore, a good manufacturing practice should be implemented during the product preparation.

Human breast milk contains commensal lactic acid bacteria (LAB) and Bifidobacterium . Several reports have suggested that human milk is a direct source of commensal bacteria found in the infant gut, and some commensal LAB and Bifidobacterium in human milk may bring positive effects to the health of breast-fed babies by shaping their gut microbiota. Therefore, researching on LAB and Bifidobacterium in breast milk is important. A total of 25 human breast milk samples were collected in Huhhot city of Inner Mongolia. The bacterial DNA of all samples was extracted, and the target sequences of LAB and Bifidobacterium were amplified using specific primers. The PCR products were sequenced by Pacific Biosciences (PacBio) single-molecule, real-time sequencing technology (SMRT). Then the diversity and composition of LAB and Bifidobacterium were analyzed at species level using bioinformatic methods. We found 120 species of LAB including Lactobacillus delbrueckii , Lactobacillus plantarum , Lactobacillus gallinarum , Lactobacillus fermentum , Staphylococcus epidermidis , Streptococcus parauberis , Streptococcus mitis , Streptococcus salivarius and so on, as well as 11 species of Bifidobacterium including Bifidobacterium catenulatum , Bifidobacterium longum , Bifidobacterium stercoris and Bifidobacterium bifidum and so on. Among them, the species Lactobacillus delbrueckii , Lactobacillus plantarum , Lactobacillus gallinarum , Lactobacillus fermentum , Lactobacillus rhamnosus and Streptococcus salivarius were detected in all samples. Additionally, almost all of the detected Bifidobacterium species were present in all samples, except Bifidobacterium pseudolongum and Bifidobacterium mongoliense and. The lactation stage had a significant effect on the diversity and composition of LAB but not Bifidobacterium . Results from PERMANOVA based on Bray-Curtis distances further confirmed the significant effect of lactation stage on LAB structure in human milk ( P 0.05). To compare the structure of the LAB community among three milk groups (i.e. colostrum, less than 5 d after delivery; transitional milk, 5–14 d after delivery; mature milk, more than 15 d after delivery), we performed Bray-Curtis PCoA based on OTU distribution and abundance. The PCoA results suggested apparent LAB structural differences among the three groups, as the symbols representing the individual groups were separated on the score plot with only minor overlap. In addition, the relative abundances of Lactobacillus delbrueckii ( P 0.01) and Lactobacillus plantarum ( P 0.05) in colostrum and mature milk were significantly higher than that of the transitional milk. Significantly more Lactobacillus rhamnosus was found in colostrum than in transition milk and mature milk ( P 0.05), whereas, the Lactobacillus helveticus in transition milk was remarkably higher than another two stages human milk ( P 0.05). However, there was no difference in the distribution of Bifidobacterium species among human milk of three lactation stages. The breast milk from the sampled mothers in Inner Mongolia could be divided into three subtypes according to the LAB composition: the first subtype contained high proportion of Lactobacillus delbrueckii (>50%); the second subtype had less than 50% of Lactobacillus delbrueckii together with more Lactobacillus plantarum and other unclassified LAB species; the third subtype contained more than 5% of Lactobacillus gallinarum . Interestingly, the three subtypes were largely consistent with the grouping by lactation stage except for the colostrum. Nevertheless, most colostrum samples belonged to the first and second subtypes. We also found significant correlations between some LAB and Bifidobacterium . In recent years, we have started isolating probiotics from human breast milk in order to identify suitable probiotic bacteria in breast milk of the Chinese population. This study has established a new method to assess the diversity of LAB and Bifidobacterium , which will serve as reference and guiding protocol for screening probiotics in human breast milk.

Abstract. Wulansari PD, Rahayu N, Kusuma RJ, Sukarno AS. 2023. Short Communication: Diversity of indigenous LAB from kefir grains cultured in goat milk based on phenotypic characteristics for probiotic candidates. Biodiversitas 24: 6389-6395. Kefir grain is the starter of kefir products containing the symbiotic compound of lactic acid bacteria (LAB) and yeast. Microbial diversity in kefir grain is affected by multiple factors that include the origin of microbiota, upkeep, storage, and types of milk. Goat milk exhibits various superior properties in composition or characteristics. These have inspired the present study to obtain the local isolates from LAB derived from native Indonesian kefir grain produced and revitalized using goat milk. This study aimed to obtain local LAB isolates from kefir grain through phenotypic identification and characterization. The morphology of phenotypic identification consisted of macroscopic, microscopic, and biochemical identification. Based on macroscopic and microscopic observation, the isolates were milky white, circular, entire edge, smooth and shiny surface with convex elevation, coccus/basil shaped, Gram-positive, negative catalase, non-motile, non-spore, non-producing CO2, heterofermentative/homofermentative, able to ferment various carbohydrates, growing at 37 and 45°C, pH 4.4, and salinity level of 6.5%. A scientific approach enabled to obtaining of several LAB strains, namely nine isolates of Lactococcus and seven isolates of Lactobacillus. It takes genotypic identification to promote the identification process up to the species level.

Since little is known about the genetic diversity of lactic acid bacteria (LAB) isolates from the fermentation pit mud (FPM), we sought to evaluate the bacterial structure, identify the LAB isolates and investigate the genotype and genetic diversity of the LAB isolates. Using high-throughput MiSeq sequencing, we identified seven dominant bacterial genera in FPM. Lactobacillus had the highest abundance. We isolated 55 LAB strains. These isolates were all identified as Lacticaseibacillus paracasei. Using an extant multilocus sequence typing (MLST) scheme, isolates were assigned to 18 sequence types (STs) and three clonal complexes. ST1, the largest group, mainly comprised FPM isolates. Niche-specific ST2 to ST18 only contained FPM isolates. Isolates could be divided into four lineages, with most assigned to Lineage 1. Only one FPM isolate was classified as L. paracasei subsp. paracasei. Other isolates could not be classified at the subspecies level using the seven MLST loci. Lactobacilli account for a high proportion of bacteria in pit mud. Based on the traditional culture method, L. paracasei was the dominant species, and these isolates exhibit a high ethanol tolerance, high intraspecific diversity and specific genetic profiles. The study described the characterization of FPM bacterial diversity, giving an insight into the genetic diversity of L. paracasei strains present in FPM.