Survival and Activity of Probiotic Lactobacilli in Skim Milk Containing Prebiotics

ABSTRACT: Seven strains were cultured anaerobically at 37 °C for 48 h in 12% (w/v) reconstituted skim milk containing 5% (w/v) Hi-maize, lactulose, inulin, or raftilose. Their viability was determined before and after 4 wk of storage at 4 °C. Doubling time (Td) was also determined. Concentrations of acetic and lactic acids produced during fermentation and storage were determined. The Td ranged from 301 to 751 min. In general, the viability of lactobacilli after storage was greatest with inulin. The pH after storage in skim milk ranged from 4.34 (for ASCC 1520 with raftilose) to 4.10 (for ATCC 15820 with inulin). Survival of lactobacilli in prebiotic was strainspecific but in general their survival was enhanced.     

Growth, Viability and Activity of Bifidobacterium spp. in Skim Milk Containing Prebiotics

Growth, activity and mean doubling time (T_d) of five Bifidobacterium species in the presence of four types of prebiotics, and concentrations of acetic and lactic acids were determinted during fermentation and after 4 weeks of refrigerated storage. The T_d was lowest for B. animalis with raftilose and inulin. Retention of viability of bifidobacteria was greatest with hi‐amylose corn starch (hi‐maize). The average pH of skim milk at the end of 4 wks storage averaged 4.34 (for B. animalis with raftilose) to 4.07 (for B. longum with inulin). The highest levels of acetic acid and lactic acid were produced by B. pseudolongum with lactulose and B. infantis with lactulose, respectively.     

Vitamin profiles of kefirs made from milks of different species

Kefirs were produced under laboratory conditions using 10 different kefir grain cultures from different households, and reconstituted cow, ewe, goat and mare milk as substrates. The base milks and corresponding kefirs were examined for their content of water-soluble vitamins and orotic acid. On average, enrichment of vitamin concentration of>20% was observed with thiamine (only ewe milk kefirs), pyridoxine (kefirs from all milk species except cow milk), and folic acid (kefirs from all milk species except mare milk). Orotic acid content was reduced during fermentation throughout.     

Microbiological and chemical properties of kefir manufactured by entrapped microorganisms isolated from kefir grains

In this study, various yeasts (Kluyveromyces marxianus, Saccharomyces turicensis, Pichia fermentans) and lactic acid bacteria (Lactobacillus kefiranofaciens, Lactobacillus kefiri, Leuconostoc mesenteroides) were entrapped in 2 different microspheres using an entrapment ratio for the strains that was based on the distribution ratio of these organisms in kefir grains. The purpose of this study was to develop a new technique to produce kefir using immobilized starter cultures isolated from kefir grains. An increase in cell counts with fermentation cycles was observed for both the lactic acid bacteria (LAB) and yeasts, whereas the cell counts of kefir grains were very stable during cultivation. Scanning electron microscopy showed that the short-chain lactobacilli and lactococci occupied the surface of the LAB microspheres, whereas the long-chain lactobacilli were inside the microspheres. When the yeasts were analyzed, cells at a high density were entrapped in cracks on the surface and within the microspheres, where they were surrounded by the short-chain lactobacilli. The distribution of the LAB and yeast species in kefir produced from grains and microspheres showed that there was no significant difference between the kefirs produced by the 2 methods; moreover, Leu. mesenteroides and K. marxianus were the predominating microflora in both types of kefir. There was no significant difference in the ethanol and exopolysaccharide contents between the 2 kefirs, although the acidity was different.     

Organic acids and volatile flavor components evolved during refrigerated storage of kefir

Kefir samples were prepared and transferred to sterile jars for storage at 4 degrees C. After 0, 7, 14, and 21 d of storage, the pH, organic acid, and volatile flavor component content were determined to monitor possible flavor changes during storage. Stored samples were analyzed for organic acid (orotic, citric, pyruvic, lactic, uric, acetic, propionic, butyric, and hippuric) content by HPLC with UV detection at 275 nm. Acetoin, ethanol, acetaldehyde, and diacetyl were monitored using gas chromatography equipped with a headspace autosampler. There was no significant decrease in average pH of samples between d 0 and 21 of storage (P>0.05). Lactic acid concentration increased during storage, reaching a maximum of 7739 ppm by d 21. Orotic and citric acids increased slightly during storage. Although pyruvic and hippuric acids are produced during fermentation, neither was detected during storage. Acetic, propionic, and butyric acids were not detected during kefir storage. Ethanol concentrations increased during storage and reached 0.08% by d 21. The amounts of acetaldehyde and acetoin, common flavor substances in many cultured dairy products, increased during fermentation. Acetaldehyde content in kefir samples doubled from d 0 to 21, reaching a final concentration of 11 microg/g. During storage, the concentration of acetoin decreased from 25 ppm on d 0 to 16 ppm on d 21. However, diacetyl, another common flavor component in cultured dairy products, was not detected during fermentation or storage.     

Comparison of antioxidant capacity of cow and ewe milk kefirs

This research aimed to evaluate the effects of using either grain or commercial starter culture on the antioxidative capacity of cow and ewe milk kefirs. The antioxidant capacity of kefir samples during fermentation and 21 d of storage was assessed by using 3 assays: 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) radical cation decolorization; 2,2-diphenyl-1-picrylhydrazyl (DPPH^?) radical scavenging activity assay; and Fe⁺³-reducing power (ferric reducing antioxidant power assay, FRAP). Vitamin E and β-carotene contents were also quantified. All kefir samples exhibited varying values for DPPH, ABTS, and FRAP assays depending on the starter culture and milk type. Vitamin E and β-carotene contents were similar in all kefir samples during storage. The results of this study suggest that milk type (cow or ewe) and culture type (kefir grains or commercial starter) were the significant parameters for the antioxidative activity of kefir.     

Rheological,texture and sensory properties of kefir with high performance and native inulin

Rheological, textural, and sensory properties of kefirs with inulin were analysed. Besides, the effect of skim milk powder (SMP) and milk fat substitution was examined by high performance (HP) and native inulin. One-third of SMP was substituted by native (IQ) or HP (TEX!, HPX) inulin. Hardness of kefir with IQ inulin was lower than that with HP inulins or pure SMP. Analysis of cohesiveness and adhesiveness showed that kefir with inulin had higher firmness. Rheological analysis revealed that all manufactured kefirs had higher storage than loss modulus, and exhibited thixotropic and shear thinning behaviour. There were no significant differences in odour and consistency between kefirs with and without inulin. Only in case of whole milk powder (WMP) kefir, panellists noticed the difference in flavour, although kefirs based on SMP had the same scores. Substitution of SMP by inulin allowed for reducing 15% and 37% of caloric value in comparison with SMP and WMP kefir, respectively.     

Kefir Immobilized on Corn Grains as Biocatalyst for Lactic Acid Fermentation and Sourdough Bread Making

Abstract: The natural mixed culture kefir was immobilized on boiled corn grains to produce an efficient biocatalyst for lactic acid fermentation with direct applications in food production, such as sourdough bread making. The immobilized biocatalyst was initially evaluated for its efficiency for lactic acid production by fermentation of cheese whey at various temperatures. The immobilized cells increased the fermentation rate and enhanced lactic acid production compared to free kefir cells. Maximum lactic acid yield (68.8 g/100 g) and lactic acid productivity (12.6 g/L per day) were obtained during fermentation by immobilized cells at 37 °C. The immobilized biocatalyst was then assessed as culture for sourdough bread making. The produced sourdough breads had satisfactory specific loaf volumes and good sensory characteristics. Specifically, bread made by addition of 60% w/w sourdough containing kefir immobilized on corn was more resistant regarding mould spoilage (appearance during the 11^th day), probably due to higher lactic acid produced (2.86 g/Kg of bread) compared to the control samples. The sourdough breads made with the immobilized biocatalyst had aroma profiles similar to that of the control samples as shown by headspace SPME GC‐MS analysis.     

Viability of microencapsulated Bifidobacterium animalis ssp. lactis BB12 in kefir during refrigerated storage

The viability of free and immobilized cells of Bifidobacterium animalis ssp. lactis BB12 incorporated into kefir was studied for 28 days during refrigerated storage. The immobilized bifidobacteria were added directly to previously prepared kefir. Titratable acidity, pH, ethanol, fat, protein and lactose were evaluated in the kefir with bifidobacteria after the storage. The survival of the free and microencapsulated bifidobacteria was evaluated during the storage period and in simulated gastric juice. The pH of kefirs ranged from 4.3 to 4.6. Encapsulation improved significantly the survival of bifidobacteria during exposure to nisin, during the storage period and in simulated gastric juice.     

Amino acid profiles of lactic acid bacteria, isolated from kefir grains and kefir starter made from them

The characteristics of cell growth, lactic acid production, amino acid release and consumption by single-strain cultures of lactic acid bacteria (isolated from kefir grains), and by a multiple-strain kefir starter prepared from them, were studied. The change in the levels of free amino acids was followed throughout the kefir process: single-strain kefir bacteria and the kefir starter (Lactococcus lactis C15-1%+Lactobacillus helveticus MP12-3%+(Streptococcus thermophilus T15+Lactobacillus bulgaricus HP1 = 1:1)-3%) were cultivated in pasteurized (92 degrees C for 20 min) cow's milk (3% fat content) at 28 degrees C for 5 h (the kefir starter reached pH 4.7) and subsequently grown at 20 degrees C for 16 h; storage was at 4 degrees C for 168 h. The strain L. helveticus MP12 was unrivaled with respect to free amino acid production (53.38 mg (100 g)(-1)) and cell growth (17.8 x 10(8) CFU ml(-1)); however, it manifested the lowest acidification activity. L. bulgaricus HP1 released approximately 3.7 times less amino acids, nearly 5 times lower cell growth, and produced about 1.2 times more lactic acid. S. thermophilus T15 demonstrated dramatically complex amino acid necessities for growth and metabolism. With L. lactis C15, the highest levels of growth and lactic acid synthesis were recorded (18.3 x 10(8) CFU ml(-1) and 7.8 g l(-1) lactic acid at the 21st hour), and as for free amino acid production, it approximated L. bulgaricus HP1 (17.03 mg (100 g)(-1) maximum concentration). In the L. lactis C15 culture, the amino acids were used more actively throughout the first exponential growth phase (by the 10th hour) than during the second growth phase. The unique properties of the L. helveticus MP12 strain to produce amino acids were employed to create a symbiotic bioconsortium kefir culture, which, under conditions of kefir formation, enhanced lactic acid production and shortened the time required to reach pH 4.7; intensified cell growth activity, resulting in a respective 90- and 60-fold increase in the concentration of lactobacilli and cocci in the mixed culture compared to individual cultures; and accumulated free amino acids in the final kefir with higher total concentrations (56.88 mg (100 g)(-1)) and an individual concentration of essential amino acids (1.5 times) greater than that of yogurt.     

Comparative study of the biochemical changes and volatile compound formations during the production of novel whey-based kefir beverages and traditional milk kefir

Cheese whey (CW) and deproteinised cheese whey (DCW) were investigated for their suitability as novel substrates for the production of kefir-like beverages. Lactose consumption, ethanol production, as well as organic acids and volatile compounds formation, were determined during CW and DCW fermentation by kefir grains and compared with values obtained during the production of traditional milk kefir. The results showed that kefir grains were able to utilise lactose from CW and DCW and produce similar amounts of ethanol (7.8–8.3 g/l), lactic acid (5.0 g/l) and acetic acid (0.7 g/l) to those obtained during milk fermentation. In addition, the concentration of higher alcohols (2-methyl-1-butanol, 3-methyl-1-butanol, 1-hexanol, 2-methyl-1-propanol, and 1-propanol), ester (ethyl acetate) and aldehyde (acetaldehyde) in cheese whey-based kefir and milk kefir beverages were also produced in similar amounts. Cheese whey and deproteinised cheese whey may therefore serve as substrates for the production of kefir-like beverages similar to milk kefir.     

Phenolic content and antioxidant activity of donkey milk kefir fortified with sulla honey and rosemary essential oil during refrigerated storage

The aim of this work was to evaluate the phenolic content and antioxidant activity of donkey kefir fortified with sulla honey and rosemary essential oil, during refrigerated storage. The type of flavouring strongly influenced the antioxidant activity of the kefir: rosemary essential oil kefir showed the highest 2,2′‐azino‐bis‐(3‐ethylbenzthiazoline‐6‐sulfonic acid values; sulla honey kefir showed the highest ferric‐reducing antioxidant power values but, at the same time, in both fortified kefirs, the thiol content decreased, probably because of the formation of polyphenol‐protein complexes that would have influence the availability of bioactive components. The antioxidant activity increased significantly during refrigerated storage, showing the highest values after 15 days. Sensory analysis highlighted the fact that the donkey kefir was well accepted by consumers. Addition of sulla honey resulted in the highest acceptability, while addition of rosemary essential oil kefir was less accepted by consumers. This knowledge provides a basis that could lead to the production of fortified donkey kefir with specific nutraceutical characteristics.     

Inhibitory activity of cheese whey fermented with kefir grains

We investigated the chemical and microbiological compositions of three types of whey to be used for kefir fermentation as well as the inhibitory capacity of their subsequent fermentation products against 100 Salmonella sp. and 100 Escherichia coli pathogenic isolates. All the wheys after fermentation with 10% (wt/vol) kefir grains showed inhibition against all 200 isolates. The content of lactic acid bacteria in fermented whey ranged from 1.04 × 10(7) to 1.17 × 10(7) CFU/ml and the level of yeasts from 2.05 × 10(6) to 4.23 × 10(6) CFU/ml. The main changes in the chemical composition during fermentation were a decrease in lactose content by 41 to 48% along with a corresponding lactic acid production to a final level of 0.84 to 1.20% of the total reaction products. The MIC was a 30% dilution of the fermentation products for most of the isolates, while the MBC varied between 40 and 70%, depending on the isolate. The pathogenic isolates Salmonella enterica serovar Enteritidis 2713 and E. coli 2710 in the fermented whey lost their viability after 2 to 7 h of incubation. When pathogens were deliberately inoculated into whey before fermentation, the CFU were reduced by 2 log cycles for E. coli and 4 log cycles for Salmonella sp. after 24 h of incubation. The inhibition was mainly related to lactic acid production. This work demonstrated the possibility of using kefir grains to ferment an industrial by-product in order to obtain a natural acidic preparation with strong bacterial inhibitory properties that also contains potentially probiotic microorganisms.     

The microbial diversity of water kefir

The microbial diversity of water kefir, made from a mixture of water, dried figs, a slice of lemon and sucrose was studied. The microbial consortia residing in the granules of three water kefirs of different origins were analyzed. A collection of 453 bacterial isolates was obtained on different selective/differential media. Bacterial isolates were grouped with randomly amplified polymorphic DNA (RAPD)-PCR analyses. One representative of each RAPD genotype was identified by comparative 16S rDNA gene sequencing. The predominant genus in water kefirs I and II was Lactobacillus, which accounted for 82.1% in water kefir I and 72.1% in water kefir II of the bacterial isolates. The most abundant species in water kefirs I and II were Lactobacillus hordei and Lb. nagelii followed by considerably lower numbers of Lb. casei. Other lactic acid bacteria (LAB) were identified as Leuconostoc mesenteroides and Lc. citreum in all three water kefirs. The most abundant species in water kefir III was Lc. mesenteroides (28%) and Lc. citreum (24.3%). A total of 57 LAB belonging to the species of Lb. casei, Lb. hordei, Lb. nagelii, Lb. hilgardii and Lc. mesenteroides were able to produce exopolysacchrides from sucrose. Non LABs were identified as Acetobacter fabarum and Ac. orientalis. The Acetobacter species were more prevalent in consortium III. Cluster analyses of RAPD-PCR patterns revealed an interspecies diversity among the Lactobacillus and Acetobacter strains. Aditionally, Saccharomyces cerevisiae, Lachancea fermentati, Hanseniaospora valbyensis and Zygotorulaspora florentina were isolated and identified by comparison of partial 26S rDNA sequences and FTIR spectroscopy.     

Influence of adjunct cultures on angiotensin‐converting enzyme (ACE)‐inhibitory activity,organic acid content and peptide profile of kefir

The angiotensin‐converting enzyme (ACE)‐inhibitory activities, peptide profiles and organic acid contents in kefir produced by kefir grains plus lactic acid bacteria as adjunct cultures were determined. All the kefir samples showed almost similar peptide profiles as detected by RP‐HPLC, but quantitative differences were observed during storage. The ACE‐inhibitory activities of different lactic cultures did not exhibit a linear tendency during storage period. After 7 days of storage, there was a significant increase in ACE‐inhibitory activity of the sample fermented with Lactobacillus helveticus. However, a kefir sample containing Streptococcus thermophilus, Lactobacillus acidophilus and Bifidobacterium animalis subsp. lactis exhibited a higher ACE‐inhibitory activity (92.23%) compared to the other samples.     

Turkish kefir and kefir grains: microbial enumeration and electron microscobic observation†

Microbial populations in kefir and kefir grains were enumerated by plating. Total lactic acid bacteria, lactoccocci, lactobacilli and yeast populations increased during fermentation and increased slightly during cold storage. Kefir grains had a lactic acid bacteria : yeast ratio of 10 ⁹  : 10 ⁶ . In further studies, kefir grains were observed using scanning electron microscopy (SEM) methods, which indicated yeast colonization on the surface and middle part of the kefir grain. Three types of lactobacilli (short, long and curved) were noted throughout the grain. Lactococci were not observed under SEM; preparation of kefir grains for SEM may have caused removal of lactococci from the grains.     

Assessment of beverages made from milk,soya milk and whey using Iranian kefir starter culture

Kefir starter culture was used for the production of beverages, and some chemical and microbiological parameter changes were determined during 24 h of fermentation and then after 2, 7, 14, 21 and 28 days of storage at 4 °C. Three different substrates (milk, whey and soya milk) were used as fermentation media for the kefir starter culture. After the fermentation, the carbohydrate content and pH decreased, but the dry matter and fat content of the beverages were not significantly different from their substrates. During storage, lactic acid bacteria in the beverages decreased, while yeasts increased and carbohydrate, ethanol and pH changed significantly in the three beverages.     

Effect of prebiotic carbohydrates on the growth and tolerance of Lactobacillus

Resistance to gastrointestinal conditions is a requirement for bacteria to be considered probiotics. In this work, we tested the resistance of six different Lactobacillus strains and the effect of carbon source to four different gastrointestinal conditions: presence of α-amylase, pancreatin, bile extract and low pH. Novel galactooligosaccharides synthesized from lactulose (GOS-Lu) as well as commercial galactooligosaccharides synthesized from lactose (GOS-La) and lactulose were used as carbon sources and compared with glucose. In general, all strains grew in all carbon sources, although after 24 h of fermentation the population of all Lactobacillus strains was higher for both types of GOS than for glucose and lactulose. No differences were found among GOS-Lu and GOS-La. α-amylase and pancreatin resistance was retained at all times for all strains. However, a dependence on carbon source and Lactobacillus strain was observed for bile extract and low pH resistance. High hydrophobicity was found for all strains with GOS-Lu when compared with other carbon sources. However, concentrations of lactic and acetic acids were higher in glucose and lactulose than GOS-Lu and GOS-La. These results show that the resistance to gastrointestinal conditions and hydrophobicity is directly related with the carbon source and Lactobacillus strains. In this sense, the use of prebiotics as GOS and lactulose could be an excellent alternative to monosaccharides to support growth of probiotic Lactobacillus strains and improve their survival through the gastrointestinal tract.     

Influence of Galactooligosaccharides and Modified Waxy Maize Starch on Some Attributes of Yogurt

Abstract: This study examined the influence of galactooligosaccharides (GOS) and modified waxy maize starch (MWMS) addition on the growth of starter cultures, and syneresis and firmness of low‐fat yogurt during storage for 28 d at 4 °C. The control yogurt (CY) was prepared without any prebiotics. Incorporation of 2.0% (w/v) GOS improved the growth of L. delbrueckii ssp. bulgaricus ATCC 11842 resulting in a shorter fermentation time. There was a significant (P < 0.05) increase in proteolysis in yogurt made with GOS (GOSY) as measured by absorbance value (0.728). Addition of GOS resulted in higher (P < 0.05) concentration of lactic and acetic acids in comparison with that of MWMSY and the CY up to day 14, thereafter, the product showed a decrease in lactic acid content in all 3 batches until the end of storage. The level of syneresis was the lowest (2.14%) in MWMSY as compared with that of GOSY (2.35%) and CY (2.53%). There was no statistically significant (P > 0.05) difference in the firmness among the 3 types of yogurt.