High-throughput sequence-based analysis of the bacterial composition of kefir and an associated kefir grain

Lacticin 3147 is a two-peptide broad spectrum lantibiotic produced by Lactococcus lactis DPC3147 shown to inhibit a number of clinically relevant Gram-positive pathogens. Initially isolated from an Irish kefir grain, lacticin 3147 is one of the most extensively studied lantibiotics to date. In this study, the bacterial diversity of the Irish kefir grain from which L. lactis DPC3147 was originally isolated was for the first time investigated using a high-throughput parallel sequencing strategy. A total of 17 416 unique V4 variable regions of the 16S rRNA gene were analysed from both the kefir starter grain and its derivative kefir-fermented milk. Firmicutes (which includes the lactic acid bacteria) was the dominant phylum accounting for > 92% of sequences. Within the Firmicutes, dramatic differences in abundance were observed when the starter grain and kefir milk fermentate were compared. The kefir grain-associated bacterial community was largely composed of the Lactobacillaceae family while Streptococcaceae (primarily Lactococcus spp.) was the dominant family within the kefir milk fermentate. Sequencing data confirmed previous findings that the microbiota of kefir milk and the starter grain are quite different while at the same time, establishing that the microbial diversity of the starter grain is not uniform with a greater level of diversity associated with the interior kefir starter grain compared with the exterior.     

Production,characterization and use of expendable kefir starter culture in the kefir production process

Upon sterilization, freeze-dried kefir grains are to be supplemented with yeast preparation, yeast showing the lowest survival in the course of freeze drying. A mixture of 20% sucrose solution and starch appeared to be the most efficient dispersing agent to protect yeast in the freeze-drying process. A starter which is produced with the kefir starter culture, does not differ in its microbiological, biochemical and organoleptic properties from a starter as obtained with kefir grains in the same time, such starter shows a better consistency. The preparation of such starters is less laborious and less complicated than the preparation of starters using kefir grains.     

Thermally-dried free and immobilized kefir cells as starter culture in hard-type cheese production

In an attempt to seek for suitable dried cultures, thermally-dried kefir was employed as starter in hard-type cheese production and tested in cheeses ripened at 5, 18 and 22 °C. Both free and immobilised on casein kefir cells were used and compared to cheese made without starter culture. Cheese products made with free cells of kefir culture were characterized by longer preservation time, improved aroma, taste, texture characteristics and increased degree of openness. Volatile profiles obtained by GC/MS analysis revealed a 216% increase in total concentration of esters, organic acids, alcohols and carbonyl compounds between cheeses prepared with and without kefir culture.     

Evaluation of freeze-dried kefir coculture as starter in feta-type cheese production

The use of freeze-dried kefir coculture as a starter in the production of feta-type cheese was investigated. Maturation of the produced cheese at 4 degrees C was monitored for up to 70 days, and the effects of the starter culture, the salting method, and the ripening process on quality characteristics were studied. The use of kefir coculture as a starter led to increased lactic acid concentrations and decreased pH values in the final product associated with significantly higher conversion rates compared to salted rennet cheese. Determination of bacterial diversity at the end of the ripening process in salted kefir and rennet cheeses by denaturing gradient gel electrophoresis technology, based on both DNA and RNA analyses, suggested a potential species-specific inhibition of members of the genera Staphylococcus and Psychrobacter by kefir coculture. The main active microbial associations in salted kefir cheese appeared to be members of the genera Pseudomonas and Lactococcus, while in salted rennet cheese, Oxalobacteraceae, Janthinobacterium, Psychrobacter, and Pseudomonas species were noted. The effect of the starter culture on the production of aroma-related compounds responsible for cheese flavor was also studied by the solid-phase microextraction-gas chromatography-mass spectrometry technique. Kefir coculture also appeared to extend the shelf life of unsalted cheese. Spoilage of kefir cheese was observed on the 9th and 20th days of preservation at 10 and 5 degrees C, respectively, while spoilage in the corresponding rennet cheese was detected on the 7th and 16th days. Microbial counts during preservation of both types of unsalted cheese increased steadily and reached similar levels, with the exception of staphylococci, which were significantly lower in unsalted kefir cheese. All types of cheese produced with kefir as a starter were approved and accepted by the panel during the preliminary sensory evaluation compared to commercial feta-type cheese.     

Whey-cheese production using freeze-dried kefir culture as a starter

AIMS: The aim of the present study was to evaluate the use of a freeze-dried kefir culture in the production of a novel type of whey-cheese similar to traditional Greek Myzithra-cheese, to achieve improvement of the quality characteristics of the final product and the extension of shelf-life. METHODS AND RESULTS: The use of kefir culture as a starter led to increased lactic acid concentrations and decreased pH values in the final product compared with whey-cheese without starter culture. The effect of the starter culture on production of aroma-related compounds responsible for cheese flavour was also studied using the solid phase microextraction gas chromatography/mass spectrometry technique. Spoilage in unsalted kefir-whey-cheese was observed on the thirteenth and the twentieth day of preservation at 10 and 5 degrees C, respectively, while the corresponding times for unsalted whey-cheese preservation were 11 and 14 days. CONCLUSIONS: The cheeses produced were characterized as high-quality products during the preliminary sensory evaluation. An indication of increased preservation time was attributed to the freeze-dried kefir culture, which also seemed to suppress growth of pathogens. SIGNIFICANCE AND IMPACT OF THE STUDY: The results suggested the use of kefir culture as a means to extend the shelf-life of dairy products with reduced or no salt content.     

Exopolysaccharides produced by lactic acid bacteria of kefir grains

A Lactobacillus delbrueckii subsp. bulgaricus HP1 strain with high exopolysaccharide activity was selected from among 40 strains of lactic acid bacteria, isolated from kefir grains. By associating the Lactobacillus delbrueckii subsp. bulgaricus HP1 strain with Streptococcus thermophilus T15, Lactococcus lactis subsp. lactis C15, Lactobacillus helveticus MP12, and Sacharomyces cerevisiae A13, a kefir starter was formed. The associated cultivation of the lactobacteria and yeast had a positive effect on the exopolysaccharide activity of Lactobacillus delbrueckii subsp. bulgaricus HP1. The maximum exopolysaccharide concentration of the starter culture exceeded the one by the Lactobacillus delbrueckii subsp. bulgaricus HP1 monoculture by approximately 1.7 times, and the time needed to reach the maximum concentration (824.3 mg exopolysacharides/l) was shortened by 6 h. The monomer composition of the exopolysaccharides from the kefir starter culture was represented by glucose and galactose in a 1.0:0.94 ratio, which proves that the polymer synthesized is kefiran.     

Milk Kefir: Ultrastructure,Antimicrobial Activity and Efficacy on Aflatoxin B1 Production by <Emphasis Type="Italic">Aspergillus flavus</Emphasis>

The association of kefir microbiota was observed by electron microscopic examination. Scanning electron microscopic (SEM) observations revealed that kefir grain surface is very rough and the inner portions had scattered irregular holes on its surface. The interior of the grain comprised fibrillar materials which were interpreted as protein, lipid and a soluble polysaccharide, the kefiran complex that surrounds yeast and bacteria in the grain. Yeast was observed more clearly than bacteria on the outer portion of the grain. Transmission electron microscopic (TEM) observations of kefir revealed that the grain comprised a mixed culture of yeast and bacteria growing in close association with each other. Microbiota is dominated by budded and long-flattened yeast cells growing together with lactobacilli and lactococci bacteria. Bacterial cells with rounded ends were also observed in this mixed culture. Kefir grains, kefir suspensions, and kefiran were tested for antimicrobial activities against several bacterial and fungal species. The highest activity was obtained against Streptococcus faecalis KR6 and Fusarium graminearum CZ1. Growth of Aspergillus flavus AH3 producing for aflatoxin B1 for 10 days in broth medium supplemented with varying concentrations of kefir filtrate (%, v/v) showed that sporulation was completely inhibited at the higher concentrations of kefir filtrate (7–10%, v/v). The average values of both mycelial dry weights and aflatoxin B1 were completely inhibited at 10% (v/v). This is the first in vitro study about the antifungal characteristics of kefir against filamentous fungi which was manifested by applying its inhibitory effect on the productivity of aflatoxin B1 by A. flavus AH3.     

Experiences with the use of a starter culture in the fermentation of maize for ‘kenkey’ production in Ghana

Controlled fermentation of maize was carried out using six strains of Lactobacillus fermentum and one strain of yeast, Saccharomyces cerevisiae, isolated from traditionally fermented maize dough as starter cultures for inoculum enrichement. The fermentations were monitored by pH, acidity, microbiological analysis and taste panel evaluation of two products, kenkey and koko, prepared from the fermented doughs. The strains of L. fermentum used as starter culture dominated the microflora during fermentation and in most inoculated doughs the required pH was attained by 24 h instead of 48 h of dough fermentation. Higher contents of lactic acid bacteria and yeasts were observed in inoculated doughs at the initial stages of fermentation but the spontaneously fermented doughs attained similar lactic acid bacteria and yeasts counts by 24 h of dough fermentation. The organoleptic quality of kenkey and koko prepared from doughs fermented with starter culture for 48 h was not significantly different from the traditional products. Kenkey prepared from doughs fermented for 24 h with starter culture were found to be unacceptable by the taste panel although similarly produced koko was acceptable.The authors are with the Food Research Institute, Council for Scientific and Industrial Research, P.O Box M 20. Accra, Ghana.     

Whey valorisation: A complete and novel technology development for dairy industry starter culture production

Whey is the major by-product of the dairy industry, produced in large quantities and usually disposed off causing major environmental pollution, due to its high organic load that makes treatment cost prohibitive. This paper comprises a contribution on the valorisation of this high polluting liquid waste of the dairy industry, based on research for the production of novel dairy starter cultures using whey as raw material. Starter cultures are used for cheese ripening in order to: (i) accelerate ripening, (ii) improve quality and (iii) increase shelf-life. The developed technology involves biomass production from whey followed by thermal drying of cultures. Specifically, Kluyveromyces marxianus, Lactobacillus bulgaricus and kefir yeasts were thermally dried, and their efficiency in lactose and milk whey fermentations was studied. The most suitable culture regarding its technological properties was kefir, which was used for cheese ripening in freeze-dried and thermally dried form. Besides the reduction of production cost, which is an essential requirement for the food industry, the use of thermally dried kefir displayed several other advantages such as acceleration of ripening, increase of shelf-life, and improvement of hard-type cheese quality.     

Survival of Escherichia coli O157:H7, Listeria monocytogenes 4b and Yersinia enterocolitica O3 in different yogurt and kefir combinations as prefermentation contaminant

AIMS: To compare microbiological safety of yogurt, kefir and different combinations of yogurt and kefir samples by using three foodborne pathogenic strains (Escherichia coli O157:H7, Listeria monocytogenes 4b and Yersinia enterocolitica O3) as indicators. METHODS AND RESULTS: Fresh yogurt and kefir drinks were added to pasteurized milk at a 5% rate either separately or together, and then incubated at different temperatures (43 degrees C for yogurt and 30 degrees C for kefir), depending on appropriate growth temperature of their starter microflora. While traditional yogurt was found to be the least suppressive on the three pathogenic micro-organisms, samples obtained from two subsequent fermentation process (samples fermented at 43 degrees C for 3 h and at 30 degrees C for 21 h) were more suppressive than that of traditional kefir. There was no significant survival difference between E. coli O157:H7 and L. monocytogenes 4b in samples tested (P > 0.05), but Y. enterocolitica O3 was more susceptible than other two test strains (P < 0.05). CONCLUSIONS: The microbiological safety of the dairy product fermented at two consecutive periods was superior than that of traditional yogurt or kefir alone. SIGNIFICANCE AND IMPACT OF THE STUDY: These experiments may mimic what happens when yogurt and kefir starter micro-organisms are combined in a milk fermentation process with different time and temperature periods.     

Investigation of the instability and low water kefir grain growth during an industrial water kefir fermentation process

A poorly performing industrial water kefir production process consisting of a first fermentation process, a rest period at low temperature, and a second fermentation process was characterized to elucidate the causes of its low water kefir grain growth and instability. The frozen-stored water kefir grain inoculum was thawed and reactivated during three consecutive prefermentations before the water kefir production process was started. Freezing and thawing damaged the water kefir grains irreversibly, as their structure did not restore during the prefermentations nor the production process. The viable counts of the lactic acid bacteria and yeasts on the water kefir grains and in the liquors were as expected, whereas those of the acetic acid bacteria were high, due to the aerobic fermentation conditions. Nevertheless, the fermentations progressed slowly, which was caused by excessive substrate concentrations resulting in a high osmotic stress. Lactobacillus nagelii, Lactobacillus paracasei, Lactobacillus hilgardii, Leuconostoc mesenteroides, Bifidobacterium aquikefiri, Gluconobacter roseus/oxydans, Gluconobacter cerinus, Saccharomyces cerevisiae, and Zygotorulaspora florentina were the most prevalent microorganisms. Lb. hilgardii, the microorganism thought to be responsible for water kefir grain growth, was not found culture-dependently, which could explain the low water kefir grain growth of this industrial process.

     

Microbial Diversity of a Camembert-Type Cheese Using Freeze-Dried Tibetan Kefir Coculture as Starter Culture by Culture-Dependent and Culture-Independent Methods

The biochemical changes occurring during cheese ripening are directly and indirectly dependent on the microbial associations of starter cultures. Freeze-dried Tibetan kefir coculture was used as a starter culture in the Camembert-type cheese production for the first time. Therefore, it''s necessary to elucidate the stability, organization and identification of the dominant microbiota presented in the cheese. Bacteria and yeasts were subjected to culture-dependent on selective media and culture-independent polymerase chain reaction (PCR)-denaturing gradient gel electrophoresis (DGGE) analysis and sequencing of dominant bands to assess the microbial structure and dynamics through ripening. In further studies, kefir grains were observed using scanning electron microscopy (SEM) methods. A total of 147 bacteria and 129 yeasts were obtained from the cheese during ripening. Lactobacillus paracasei represents the most commonly identified lactic acid bacteria isolates, with 59 of a total of 147 isolates, followed by Lactococcus lactis (29 isolates). Meanwhile, Kazachstania servazzii (51 isolates) represented the mainly identified yeast isolate, followed by Saccharomyces cerevisiae (40 isolates). However, some lactic acid bacteria detected by sequence analysis of DGGE bands were not recovered by plating. The yeast S. cerevisiae and K. servazzii are described for the first time with kefir starter culture. SEM showed that the microbiota were dominated by a variety of lactobacilli (long and curved) cells growing in close association with a few yeasts in the inner portion of the grain and the short lactobacilli were observed along with yeast cells on the exterior portion. Results indicated that conventional culture method and PCR-DGGE should be combined to describe in maximal detail the microbiological composition in the cheese during ripening. The data could help in the selection of appropriate commercial starters for Camembert-type cheese.     

Assimilation of cholesterol in milk by kefir cultures

Summary Significant variations in the ability to assimilate cholesterol in milk were abserved among 6 kefir cultures. The amounts of cholesterol assimilated during 24 h of incubation and 48 h of storage ranged from 10.8 to 5.3 mg/dl of milk or from 84 to 41% of cholesterol in control milk (12.8 mg/dl).     

Kefir Grains Change Fatty Acid Profile of Milk during Fermentation and Storage

Several studies have reported that lactic acid bacteria may increase the production of free fatty acids by lipolysis of milk fat, though no studies have been found in the literature showing the effect of kefir grains on the composition of fatty acids in milk. In this study the influence of kefir grains from different origins [Rio de Janeiro (AR), Viçosa (AV) e Lavras (AD)], different time of storage, and different fat content on the fatty acid content of cow milk after fermentation was investigated. Fatty acid composition was determined by gas chromatography. Values were considered significantly different when p<0.05. The highest palmitic acid content, which is antimutagenic compost, was seen in AV grain (36.6g/100g fatty acids), which may have contributed to increasing the antimutagenic potential in fermented milk. Higher monounsaturated fatty acid (25.8g/100g fatty acids) and lower saturated fatty acid (72.7g/100g fatty acids) contents were observed in AV, when compared to other grains, due to higher Δ9-desaturase activity (0.31) that improves the nutritional quality of lipids. Higher oleic acid (25.0g/100g fatty acids) and monounsaturated fatty acid (28.2g/100g fatty acids) and lower saturated fatty acid (67.2g/100g fatty acids) contents were found in stored kefir relatively to fermented kefir leading to possible increase of antimutagenic and anticarcinogenic potential and improvement of nutritional quality of lipids in storage milk. Only high-lipidic matrix displayed increase polyunsaturated fatty acids after fermentation. These findings open up new areas of study related to optimizing desaturase activity during fermentation in order to obtaining a fermented product with higher nutritional lipid quality.     

Bacteriocin production by strain <Emphasis Type="Italic">Lactobacillus delbruecki</Emphasis>i ssp. <Emphasis Type="Italic">bulgaricus</Emphasis> BB18 during continuous prefermentation of yogurt starter culture and subsequent batch coagulation of milk

By screening for bacteriocin-producing lactic acid bacteria of 1,428 strains isolated from authentic Bulgarian dairy products, Lb. bulgaricus BB18 strain obtained from kefir grain was selected. Out of 11 yogurt starters containing Lb. bulgaricus BB18 and S. thermophilus strains resistant to bacteriocin secreted by Lb. bulgaricus BB18 a yogurt culture (S. thermophilus 11A+Lb. bulgaricus BB18) with high growth and bacteriocinogenic activity in milk was selected. Continuous (pH-stat 5.7) prefermentation processes were carried out in milk at 37 degrees C in a 2l MBR bioreactor (MBR AG, Zurich, Switzerland) with an IMCS controller for agitation speed, temperature, dissolved oxygen, CO2 and pH. Prefermented milk with pH 5.7 coagulated in a thermostat at 37 degrees C until pH 4.8-4.9. S. thermophilus 11A and Lb. bulgaricus BB18 grew independently in a continuous mode at similar and sufficiently high-dilution rates (D=1.83 h(-1)-S. thermophilus 11A; D=1.80 h(-1)-Lb. bulgaricus BB18). The yogurt cultures developed in a stream at a high-dilution rate (D=2.03-2.28 h(-1)). The progress of both processes (growth and bacteriocin production) depended on the initial ratio between the two microorganisms. The continuous prefermentation process promoted conditions for efficient fermentation and bacteriocinogenesis of the starter culture during the batch process: strong reduction of the times for bacteriocin production and coagulation of milk (to 4.5-5.0 h); high cell productivity (lactobacilli-4x10(12) CFU ml(-1), streptococci-6x10(12) CFU ml(-1)); high productivity of bacteriocins (4,500 BU ml(-1))-1.7 times higher than the bacteriocinogenic activity of the batch starter culture.     

Physicochemical and microbiological characteristics of Kulek cheese made from raw and heat-treated milk

The effect of heat treatment and commercial starter culture utilization on the physicochemical and microbiological properties of Kulek cheese made from raw milk with or without starter culture and heated milk with starter culture were investigated during ripening. Titratable acidity (TA) was the highest in cheeses made from heated milk while total solids (TS), salt, and fat were the highest in cheeses made from raw milk. The heat treatment significantly decreased the counts of coliforms and Enterobacteriaceae in cheeses. At the beginning of the ripening period, cheeses manufactured from heated milk with starter exhibited significantly higher counts of lactococci and proteolytic organisms and lower counts of lactobacilli than the other cheeses. After the first day, raw milk cheeses without starter showed higher microbiological counts than the others. In fresh cheeses, Lactococcus was the main lactic acid bacterium, with Lc. lactis lactis being predominant. Lactobacillus plantarum and Lactobacillus paracasei paracasei dominated at the later stages of the ripening.     

Investigation of microorganisms involved in kefir biofilm formation

Kefir is a natural fermentation agent composed of various microorganisms. To address the mechanism of kefir grain formation, we investigated the microbial role in forming kefir biofilms. The results showed that a biofilm could be formed in kefir-fermented milk and the biofilm forming ability reached the maximum at 13 days. The strains Kluyveromyces marxianus, Lactococcus lactis, Leuconostoc mesenteroides, Lactobacillus kefiri, Lactobacillus sunkii and Acetobacter orientalis were isolated from kefir biofilms by the streak-plate method. These microorganisms were analysed with respect to biofilm forming properties, including their surface characterisation (hydrophobicity and zeta potentials) and the microbial aggregation. The results indicated that Klu. marxianus possessed the strongest biofilm forming properties with the strongest hydrophobicity, lowest zeta potential and greatest auto-aggregation ability. When Klu. marxianus and Ac. orientalis were co-cultured with kefir LAB strains respectively, it was found that mixing Klu. marxianus with Lb. sunkii produced the highest co-aggregation ability. These results elucidated the mechanism of kefir biofilm formation and the microorganisms involved.     

Dietary influence of kefir on microbial activities in the mouse bowel

AIMS: In this work the microflora present in kefir, a fermented milk product, was studied together with the effect of kefir administration on different groups of indigenous bacteria of mouse bowel. METHODS AND RESULTS: Kefir microflora was composed of lactic acid bacteria, acetic acid bacteria and yeasts. Yeast population was composed of Saccharomyces cerevisiae, S. unisporus, Candida kefir, Kluyveromyces marxianus and K. lactis. The streptococci levels in kefir treated mice increased by 10-fold and the levels of sulfite-reducing clostridia decreased by 100-fold. The number of lactic acid bacteria increased significantly. CONCLUSIONS: The administration of kefir significantly increased the lactic acid bacteria counts in the mucosa of the bowel. Ingestion of kefir specifically lowered microbial populations of Enterobacteriaceae and clostridia. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first long-term study about the effects of the kefir administration on the intestinal microflora of mice.     

Development and evaluation of a starter culture for the industrial production of gari

Gari starter cultures (Gastat) were developed by mixing pure single strains of the organisms that ferment cassava. They were propagated and maintained as granules on dried cocoyam slurry. The cultures were tested for fermentative and acid-producing activity. The acidity produced at 30°C varied from 0.07% to 0.85% lactic acid with maximum levels occurring after 48 h. High levels of reducing sugar were produced during the first 24 h. The amounts produced were about 50% more than those from the self-inoculated cassava. The quality of the gari produced by the starter cultures was good and well accepted. The texture was similar to that produced by natural fermentation. These results highlight the possibility of using starter cultures in the large-scale production of gari.     

The influence of starter cultures on the formation of volatile compounds in dry smoked sausages

The differences between the composition of volatile substances in two specimens of dry smoked sausages produced using a standard and experimental (a mixture of propionic acid bacteria and bifidobacteria) cultures were studied by capillary gas chromatography. It was found that the experimental starter culture intensified the flavor-formation processes as compared with the standard culture. The experimental specimen had richer qualitative and quantitative compositions and displayed more intensive aroma and flavor. The contents of lactones and volatile terpenoids in the experimental specimen were much higher than in the control. The organoleptic characteristics of experimental dry smoked sausage specimen were considerably better.