Lactic Acid Bacterial Culture Research Articles

Perspective of Streptococcus thermophilus Strains as New Probiotics Derived from the Breast Milk of Indian Mothers

ABSTRACT In this study, two lactic acid bacterial cultures were isolated from human milk, and their probiotic attributes were evaluated. The 16S rRNA technique confirmed that the isolates share close homology to Streptococcus thermophilus and are designated as HN2 and HN3 strains. Both cultures showed significant tolerance, i.e. 74–92% and 66–88% survivability in acidic and bile conditions, respectively (p < 0.05). The hydrophobicity values ranged from 26–39% against hydrocarbons, auto-aggregation (40–51%), and β-galactosidase activity (43–102 U/mL) were observed, while antagonistic activity against pathogens ranged between 12–26 mm. The antibiotic profile demonstrated that the HN3 strain was more resistant compared to the HN2 strain. The antioxidant activities of supernatant and intact cells were determined using DPPH and ABTS method and found between 37–89%. The safety parameters data revealed that hemolytic reaction and mucin degradation were not detected. The findings suggested that the strains could be potentially targeted in functional foods for therapeutic applications following further in vivo studies.

Optimization of lactic acid bacterial starter culture to improve the quality and flavor characteristics of traditional Hurood

Hurood is a traditional fermented milk product prepared by traditional Mongolian techniques of fermenting raw milk, partial degreasing, heating, whey drainage, emulsification of curd, and molding. Currently, Hurood available in the market is generally prepared by small-scale enterprises at home or in open air. Therefore, lack of standardization of bacterial starter culture leads to variation in the flavor and sensory properties of Hurood from batch to batch. In this study, we aimed to assess the best starter culture combination obtained from 37 lactic acid bacterial strains isolated from traditional Hurood. The solidification state and sensory quality were used as indexes for determining the fermentation efficiency of the bacterial starter culture combinations. The yield and texture characteristics were used to determine the optimal ratio of bacterial strains in a combination and the processing conditions for traditional Hurood production. The most optimal bacterial culture combination was observed to be NF 9-3:NF 10-4:CH 3-1 in 5:4:1 ratio and in 3% amount. The most optimal whey temperature and heating-stirring temperature were observed to be 55°C to 60°C and 85°C to 90°C, respectively. Hurood prepared with the optimal combination of bacterial strains exhibited significantly enhanced sensory quality, flavor, and contents of AA and fatty acids. Therefore, the use of optimal starter culture of lactic acid bacteria could produce Hurood with significantly superior sensory qualities, making the product more acceptable to consumers.

A Comparative Study of Skimmed Milk and Cassava Flour on the Viability of Freeze-Dried Lactic Acid Bacteria as Starter Cultures for Yogurt Fermentation

The purpose of this study was to evaluate the survival rates and fermentation performance of three freeze-dried lactic acid bacterial cultures previously isolated from Ghanaian traditional fermented milk. LAB cultures, i.e., Lactobacillus delbrueckii, Lactococcus lactis and Leuconostoc mesenteroides, were frozen in the chamber of a Telstar (Lyoquest) laboratory freeze dryer for 10 h at -55 °C (as single and combined cultures) using skimmed milk and cassava flour as cryoprotectants held in plastic or glass cryovials. For viability during storage, freeze-dried LAB cultures were stored in a refrigerator (4 °C) and at room temperature (25 °C) for 4 weeks. The survival of freeze-dried cultures was determined by growth kinetics at 600 nm (OD600). The performance of freeze-dried LAB cultures after 4 weeks of storage was determined by their growth, acidification of milk during yogurt fermentation and consumer sensory evaluation of fermented milk using a nine-point hedonic scale. The survival rates for LAB ranged between 60.11% and 95.4% following freeze-drying. For single cultures, the highest survival was recorded for Lactobacillus delbrueckii (L12), whereas for combined cultures, the highest survival was observed for Lactococcus lactis (L3) combined with Lactobacillus delbrueckii (L12). The consumer acceptability results showed that yogurts produced from a combined starter culture of Lactococcus lactis and Lactobacillus delbrueckii or from a single culture of Lactococcus lactis were the most preferred products with Lactococcus lactis and Lactobacillus delbrueckii possessing high survival rates and high consumer acceptability in yogurt production. These findings are crucial and can be adopted for large-scale production and commercialization of yogurt.

Development and standardization of processing technique for ready-to-use lab fermented Kanji mix using refractance window dried black carrot powder

Black carrots are rich in bio-actives but underutilized owing to their short-term availability and perishable nature. Traditionally, black carrots have been used for the preparation of Kanji—a fermented non-dairy beverage prepared using natural fermentation by lactic acid bacteria and a few spices. This plant-based probiotic beverage has high antioxidant properties but there is a risk of contamination with pathogens due to uncontrolled fermentation during storage. To enhance the availability of this nutritious beverage throughout the year and to ensure the microbiological safety of the traditional fermented product, the present study was planned to optimize the process for controlled fermentation using freeze-dried lactic acid bacterial (LAB) culture and refractance window-dried black carrot powder. The physicochemical and microbiological profiles of LAB-fermented Kanji were analysed. The dried Kanji mix can be reconstituted into naturally fermented probiotic beverage with unique flavour and aroma along with ensured microbiological safety and enhanced commercial value.

FERMENTED FOOD PRODUCTS USING SEA BUCKTHORN FRUIT. OVERVIEW

Herbal raw materials are a unique source of biologically active substances that are of particular value in the treatment of various diseases. Sea buckthorn is one of the most famous and widespread medicinal plants, widely grown in the United States, Europe, Asia, the Russian Federation and the Republic of Belarus. Information on the phytochemical composition of fruits is quite fully covered in the scientific literature. Sea buckthorn fruits are rich in essential nutrients and biologically active substances, but remain less in demand. Thus, it is important to study and implement new ways of processing sea buckthorn fruits that can contribute to the development of new functional foods and attract the attention of consumers to these products. This review summarizes studies on the development of food products using sea buckthorn fruits and lactic acid bacterial cultures. Particular attention is paid to the process of malolactic fermentation and its influence on the content and profile of organic acids and polyphenols in the juice of sea buckthorn fruits, it is shown what changes in antioxidant properties and organoleptic characteristics occur. The role of sea buckthorn fruits and their components in the development of new probiotic dairy and non-dairy products is considered.

Rice flour powder carrying mixed starter culture of Lactiplantibacillus plantarum KU-LM173 and Pediococcus acidilactici KU-LM145 for fermented mussel, Perna viridis Linnaeus 1758.

To develop a dried rice flour powder (DP) formulation to contain a lactic acid bacterial starter culture for fermenting mussel meat (FM). Lactiplantibacillus plantarum KU-LM173 (LP), Enterococcus hirae KU-LM174 and Pediococcus acidilactici KU-LM145 (PA) were selected from commercial FMs and identified to have high acid and protease production. Mixed culture between LP, for high acid production, and PA, for the flavour, was the best for DP and had greater organoleptic properties than a single starter fermentation. The best ratio of DP for production was 1% of the mussel weight, while the highest numeric scoring of the organoleptic test between 3% and 6%. The starter culture fermentation accelerated over the natural (wild) fermentation and ended at day 3. The shelf life of the product was at least 30days at 30-35°C with no pathogens detected. The shelf life of DP at 4°C was 10weeks. DP with the best strains and long shelf life promoted safety of FM and reduced the processing time. High consumer acceptance, protease and acid production and flavour were unique product characteristics. Accelerated commercial FMs with effective DP formulation for the industrial sector may be plausible.

Fermentation of African nightshade leaves with lactic acid bacterial starter cultures

The interest in the consumption of African indigenous leafy vegetables increased in African countries, e.g. Kenya, within the last years. One example of African indigenous leafy vegetables is African nightshade (Solanum scabrum) which is nutritious, rich in proteins and micronutrients and therefore could contribute to a healthy diet. African nightshade has several agricultural advantages. However, the most important disadvantage is the fast perishability which leads to enormous post-harvest losses. In this study, we investigated the fermentation of African nightshade as a post-harvest processing method to reduce post-harvest losses. The two lactic acid bacterial starter strains Lactiplantibacillus plantarum BFE 5092 and Limosilactobacillus fermentum BFE 6620 were used to inoculate fermentations of African nightshade leaves with initial counts of 106–107 cfu/ml. Uninoculated controls were conducted for each fermentation trial. Fermentations were performed both in Kenya and in Germany. The success of the inoculated starter cultures was proven by the measurement of pH values and determination of lactic acid concentration. Lactobacilli strains dominated the microbiota of the starter inoculated samples in contrast to the non-inoculated controls. This was supported by classical culture-dependent plating on different microbiological media as well as by the culture-independent molecular biological methods denaturing gradient gel electrophoresis and 16S rRNA gene high-throughput amplicon sequencing. We could demonstrate that the use of the selected starter cultures for fermentation of African nightshade leaves led to controlled and reliable fermentations with quick acidification. Thus, controlled fermentation with appropriate starter cultures is a promising method for post-harvest treatment of African nightshade leaves.

Characterization of Buckwheat Beverages Fermented with Lactic Acid Bacterial Cultures and Bifidobacteria.

This study aimed to examine the effect of four different industrial starter cultures containing lactic acid bacteria (LAB) and bifidobacteria on selected characteristics of beverages prepared from buckwheat and stored at 4 °C for 28 days. This study included the determination of pH during fermentation and during refrigerated storage, determination of the number of LAB and bifidobacteria, and chromatographic analysis of carbohydrates. This study showed that the tested starter cultures effectively fermented the buckwheat beverage. There was a sufficient number of viable cells in the starter microflora for the obtained beverages to exhibit potential health-promoting properties. Beverages had stable pH values during refrigerated storage. The stored beverages showed changes in the content of selected carbohydrates, which indicates the constant biochemical activity of the present starter microflora. This study provides useful references on the metabolism of LAB in plant-based beverages.

Optimization of Sugar Reduction in the Formulation of Set-type Yogurt Using Pure Lactic Acid Bacterial Culture

Background: Reducing the sugar content of processed products has been claimed to be one of the most efficient strategies for decreasing sugar intake. To investigate what level of sugar reduction is accepted in set-type yogurt, we conducted a hedonic test focusing on the sensory attributes and assessed the nutritional quality of the yogurt. Methods: We prepared a total of 12 set-type yogurt samples using four sugar concentrations (viz. 0, 4, 5 and 12%). The starter culture was used as a 1:1 ratio mixture of Streptococcus thermophilus and Lactobacillus bulgaricus. The sensorial, chemical and microbiological analyses were performed. Result: From the investigation, yogurt containing at least 4 and 5% added sugar was found acceptable based on overall sensory score. On the hedonic scale, yogurt containing 12% sugar was more often described as too sweet compared with yogurt containing 4 and/or 5% sugar. On the other hand, the sweetness and aroma intensity for yogurt containing 0% sugar was judged as too low. The 4 and 5% added sugar yogurt was recorded highest with protein content 4.35 and 4.53%, respectively. The initial total viable count (TVC) of the yogurt made was found highest in 4 and 5% sugar yogurt each with » 5 log10 cfu/mL. These results indicate that consumers would accept set-type yogurts with 4 to 5% added sugar instead of 12 to 15%, but 0 to 3% sugar would be too low.

A diet containing native or fermented wheat bran does not interfere with natural microbiota of laying hens

Wheat bran (WB) is an important side product of the milling industry and can serve as dietary fiber compound for monogastric animals. The aim of this study was to evaluate the influence of native or fermented WB on the gut physiology and microbiology of laying hens. To accomplish this, 24 laying hens were fed the following diets: conventional diet without WB; 15% native WB in the diet; 15% WB fermented with Pleurotus eryngii; and 15% WB fermented with P. eryngii and a lactic acid bacterial culture. Immediately after slaughtering, digesta samples were taken from the jejunum, ileum and cecum, respectively. Total DNA was extracted and subsequently investigated with 16S DNA amplicon sequencing. Neither native nor fermented WB supplementations negatively affected the feed conversion ratio, laying performance or the relative abundances and alpha-diversity of microbiota in the intestine. Effects of WB-based diets on gut morphology were only recognized in the jejunum (reduced villum height and mucosa thickness). Likewise, WB supplementation decreased the digestibility of DM and starch. Based on these findings, it was demonstrated that different WB variants are applicable without exerting practically negative consequences on performance or on gut microbiota. Fermentation improved the digestibility/retention of dietary fat and phosphorus. However, no further beneficial effects were observed. This study also allowed a more in-depth view on the laying hens’ gut microbiome and its variation within the gut segments.

In vivo safety assessment of Lactobacillus fermentum strains, evaluation of their cholesterol-lowering ability and intestinal microbial modulation.

Despite the growing importance of probiotics apparent health benefits, an impediment to the use of new probiotic cultures is their safety. Hence there is a need to strictly examine the biosafety as well as health benefits of probiotics in in vivo model systems. In this study, two lactic acid bacterial (LAB) cultures Lactobacillus fermentum NCMR 2826 and FIX proven for their in vitro probiotic properties were investigated for their in vivo safety in Wistar rats. An acute toxicity study (14 days) with a high dose of biomass (1016 colony-forming units (CFU) mL-1 ) followed by a subchronic test for 13 weeks with oral feeding of the probiotic cultures in three different doses (107 , 108 and 1010 CFU mL-1 ) on a daily basis revealed the safety of the L. fermentum cultures. The probiotic feeding had no toxic effects on survival, body weight and food consumption with any of the dosages used throughout the treatment period. No statistically significant changes in relative organ weights and serum biochemical and hematological indices were found between the control and the probiotic fed animals. In addition to the safety attributes, the L. fermentum culture fed rats showed reduced serum cholesterol levels, macrovesicular steatosis and hepatocyte ballooning compared with control animals. Further, quantification of intestinal microbiota using real-time polymerase chain reaction (PCR) analysis from animal feces indicated a significant increase and stability of Lactobacillus and Bifidobacterium counts but a decrease of Escherichia coli numbers. This study of safety and beneficial features highlights the use of the two native L. fermentum isolates as potential probiotic food supplements. © 2019 Society of Chemical Industry.

Persistence of a mixed lactic acid bacterial starter culture during lysine fortification of sourdough breads by addition of pistachio powder

Pistachio powder was added to flour or semolina to evaluate its contribution to increase the amount of lysine in bread. Bread production was carried out by sourdough technology using a selected 3-species (Lactobacillus sanfranciscensis/Leuconostoc citreum/Weissella cibaria) lactic acid bacterial (LAB) starter culture. All sourdoughs were subjected to a long-time fermentation (21 h) and showed levels of LAB around 109 CFU/g, indicating the suitability of pistachio powder for lactic fermentation. Yeasts were also detected, in particular in semolina trials. MiSeq Illumina technology was applied to investigate the bacterial composition of sourdoughs evidencing a different distribution of LAB species among the trials with Lactobacillus as major LAB group in almost all sourdoughs. Physicochemical parameters were comparable among the trials. After baking, pistachio powder was found not to influence the height of the breads, but pistachio breads were more firm than control breads. Color of the breads, void fraction and cell density, were influenced by pistachio powder. The amount of lysine increased consistently thanks to pistachio supplementation which also determined a higher presence of o-xylene, p-cymene and limonene and the appearance of α-pinene and 1-octen-3-ol in breads. Sensory tests showed the best appreciation scores for the breads produced with flour and pistachio powder.

Isolation and characterisation of angiotensin converting enzyme (ACE) inhibitory peptides in fermented milk of malabari goat

A study was conducted to isolate and characterise the angiotensin converting enzyme (ACE) inhibitory peptides from fermented milk of Malabari goat. Goat milk was fermented with seven different lactic acid bacterial cultures including Lactobacillus acidophilus (MTCC 10307), L. plantarum (NCDC 379), L. casei (NCDC 017), L. bulgaricus (NCDC 253), L. paracasei sub sp . paracasei (NCDC 022), L. rhamnosus (MTCC 8712) and L. helveticus (NCDC 192). These cultures were inoculated at 3 and 4% levels for the preparation of fermented milk. The pH, titratable acidity, lactic acid bacterial count, proteolytic activity and ACE inhibitory activity of the fermented milk ranged from 4.07 to 5.69, 0.36 to 1.24% lactic acid, 9.79 to 10.25 log cfu/ml, 0.32 to 0.69 and 67.56 to 97.55%, respectively. The highest ACE inhibitory activity was observed in milk fermented with L. plantarum (NCDC 379). Fermented milk with high ACE inhibitory activity were subjected to reverse phase high performance liquid chromatography to isolate the ACE inhibitory peptides. Characterisation of the peptides was done by Tris-tricine SDS-PAGE. The molecular weight of ACE inhibitory peptides was 1.4 kDa. It can be concluded that Malabari goat milk fermented with lactic acid bacteria is a rich source of ACE inhibitory peptides.

MICROBIOLOGICAL INVESTIGATION OF ARTISANAL CAUCASIAN YOGURT-LIKE PRODUCT MATSONI

Aim. To investigate microbial composition of the home-made Matsoni samples and select the bacterial cultures for further development of the standardized starter(s), challenging modern biotechnological requirements. Methods. Strain isolation and purification, culture evaluation for their catalase and urease activities, casein fermentation, galactose degradation ability, exopolysaccharide (EPS) and bacteriocin production, and lysogeny. Results. One hundred forty four lactic acid bacterial cultures were isolated from thirty five homemade Matsoni samples, collected from twenty four different settlements in Georgia during years 2013–2014. Bacterial structure of the starters showed great diversity in their composition, 3% of isolates showed casein degradation and EPS production potential, 32% were positive for galactose fermentation, 19% showed urease activity, 17% revealed antagonistic properties and 14% were denoted as lysogenic. Conclusions. The developed strain collection shows potential to provide various organoleptic properties to the end products and, thus, after molecular characterization, can be easily used for construction of new standardized Matsoni starters.

The addition of citrate stimulates the production of acetoin and diacetyl by a citrate-positive Lactobacillus crustorum strain during wheat sourdough fermentation

In traditional sourdough fermentation recipes of artisan bakeries, often extra ingredients are added to the flour-water mixture. This may accentuate the aroma and taste of the baked products produced from such sourdoughs. This is made possible, for instance, by stimulating certain microbial activities during fermentation. This study examined the effects of the addition of citrate (a food-grade organic acid present in milk and lemons) on wheat sourdough fermentation in the presence of a citrate-positive, homofermentative, lactic acid bacterial starter culture strain, namely Lactobacillus crustorum LMG 23699. Both liquid and firm wheat sourdoughs were produced. The starter culture strain was able to steer all wheat sourdough fermentations performed, as it always prevailed due to its competitiveness, as shown through culture-dependent microbiological plating and culture-independent bacterial community profiling. Moreover, it possessed all enzyme-encoding genes (as unraveled through genome mining) necessary to convert citrate into desirable compounds such as lactic acid, acetic acid, succinate, acetoin, diacetyl, and 2,3-butanediol. Indeed, citrate addition to the wheat flour-water mixture had an impact on the sourdough fermentation dynamics and thus on the aroma profile of the liquid and firm sourdoughs produced and breads made thereof. A higher final pH, higher total titratable acidity values, and low yeast counts were found in wheat sourdoughs produced with citrate. In particular, the starter culture strain added converted the supplemented citrate into more l-lactic acid as well as acetoin and diacetyl (buttery aroma compounds), which was independent of the dough yield. The buttery aroma compounds were also accentuated in the concomitant breads produced. Further, organic acid production was stimulated in the sourdoughs, whereas increased pyrazine concentrations occurred in the breads. Consequently, citrate supplementation to wheat sourdoughs could be of interest to produce baked goods with enhanced buttery aroma compounds and notes.

Antimicrobial activity of cyclic dipeptides produced by Lactobacillus plantarum LBP-K10 against multidrug-resistant bacteria, pathogenic fungi, and influenza A virus

The antimicrobial activity of lactic acid bacteria (LAB) is well recognized, particularly because they can produce antimicrobial cyclic dipeptides (CDPs). CDPs serve as attractive scaffolds for drug design owing to the structural variety of their chiral side chains. Using filtrates from fermented kimchi produced using Leuconostoc mesenteroides LBP-K06 starter cultures, we previously demonstrated a combined chromatographic fractionation and gas chromatography-mass spectroscopy method for screening bioactive CDPs. However, the entire pool of CDPs from a given lactic acid bacterial culture is not fully understood. The present study identified the CDP produced by Lactobacillus plantarum LBP-K10, which was the most potent antimicrobial isolated from Korean kimchi. In total, 15 CDPs containing proline and a single CDP without proline were identified in the culture filtrates (CFs) using gas chromatography-mass spectroscopy (GC-MS). These CDPs were also commonly found in the CFs from other isolated LAB. We developed a new method for purifying a massive amount of complete sets of CDPs from CFs using the anion exchange resins Amberlite IRA-67, Purolite A420S, or both. Compared with the activities of the individual CDPs, the entire set showed remarkable potency including bioactivities against multidrug-resistant Gram-positive and Gram-negative bacteria, human and plant pathogenic fungi, and the influenza A virus (H3N2). We demonstrated that the combination of 16 CDPs naturally produced by one organism exhibited strong antimicrobial activity. This novel strategy for profiling metabolites would enable more effective assessments of fermented foods and LAB.

Potential of a lactic acid bacterial starter culture with gamma-aminobutyric acid (GABA) activity for production of fermented sausage.

The ability of lactic acid bacterial starter cultures to produce gamma-aminobutyric acid (GABA) during sausage fermentation was studied. Among 305 strains of lactic acid bacteria isolated from kimchi samples, 11 strains were selected as starter candidates based on the following criteria: growth speed, pH lowering ability, and biogenic amine productivity including GABA-producing activity. During in vitro tests, the Y8 (Lactobacillus brevis), O52, and KA20 strains produced 39.00±1.36, 49.73±3.80, and 64.59±0.61mg/kg of GABA, respectively. Interestingly, although isolate Y8 showed low productivity in vitro, the GABA content it produced during in situ tests (61.30±2.61mg/kg) was similar to that produced by isolate PM3 (L. brevis) used as positive control (69.64±2.20mg/kg). Therefore, isolate Y8 was selected as the best functional starter culture for the production of fermented sausage because it exhibited rapid growth, safety, and abundant GABA productivity.

Proline-Based Cyclic Dipeptides from Korean Fermented Vegetable Kimchi and from Leuconostoc mesenteroides LBP-K06 Have Activities against Multidrug-Resistant Bacteria.

Lactobacillus plantarum and Leuconostoc mesenteroides play a prominent role as functional starters and predominant isolates in the production of various types of antimicrobial compound-containing fermented foods, especially including kimchi. In the case of the bioactive cyclic dipeptides, their racemic diastereomers inhibitory to bacteria and fungi have been suggested to come solely from Lactobacillus spp. of these strains. We previously demonstrated the antifungal and antiviral activities of proline-based cyclic dipeptides, which were fractionated from culture filtrates of Lb. plantarum LBP-K10 originated from kimchi. However, cyclic dipeptides have not been identified in the filtrates, either from cultures or fermented subject matter, driven by Ln. mesenteroides, which have been widely used as starter cultures for kimchi fermentation. Most importantly, the experimental verification of cyclic dipeptide-content changes during kimchi fermentation have also not been elucidated. Herein, the antibacterial fractions, including cyclo(Leu-Pro) and cyclo(Phe-Pro), from Ln. mesenteroides LBP-K06 culture filtrates, which exhibited a typical chromatographic retention behavior (tR), were identified by using semi-preparative high-performance liquid chromatography and gas chromatography-mass spectrometry. Based on this finding, the proline-based cyclic dipeptides, including cyclo(Ser-Pro), cyclo(Tyr-Pro), and cyclo(Leu-Pro), were additionally identified in the filtrates only when fermenting Chinese cabbage produced with Ln. mesenteroides LBP-K06 starter cultures. The detection and isolation of cyclic dipeptides solely in controlled fermented cabbage were conducted under the control of fermentation-process parameters concomitantly with strong CDP selectivity by using a two-consecutive-purification strategy. Interestingly, cyclic dipeptides in the filtrates, when using this strain as a starter, increased with fermentation time. However, no cyclic dipeptides were observed in the filtrates of other fermented products, including other types of kimchi and fermented materials of plant and animal origin. This is the first report to conclusively demonstrate evidence for the existence of antimicrobial cyclic dipeptides produced by Ln. mesenteroides in kimchi. Through filtrates from lactic acid bacterial cultures and from fermented foods, we have also proved a method of combining chromatographic fractionation and mass spectrometry-based analysis for screening cyclic dipeptide profiling, which may allow evaluation of the fermented dairy foods from a new perspective.

Heme Derived from Corynebacterium glutamicum: A Potential Iron Additive for Swine and an Electron Carrier Additive for Lactic Acid Bacterial Culture.

To investigate the potential applications of bacterial heme, aminolevulinic acid synthase (HemA) was expressed in a Corynebacterium glutamicum HA strain that had been adaptively evolved against oxidative stress. The red pigment from the constructed strain was extracted and it exhibited the typical heme absorbance at 408 nm from the spectrum. To investigate the potential of this strain as an iron additive for swine, a prototype feed additive was manufactured in pilot scale by culturing the strain in a 5 ton fermenter followed by spray-drying the biomass with flour as an excipient (biomass: flour = 1:10 (w/w)). The 10% prototype additive along with regular feed was supplied to a pig, resulting in a 1.1 kg greater increase in weight gain with no diarrhea in 3 weeks as compared with that in a control pig that was fed an additive containing only flour. To verify if C. glutamicum-synthesized heme is a potential electron carrier, lactic acid bacteria were cultured under aerobic conditions with the extracted heme. The biomasses of the aerobically grown Lactococcus lactis, Lactobacillus rhamosus, and Lactobacillus casei were 97%, 15%, and 4% greater, respectively, than those under fermentative growth conditions. As a potential preservative, cultures of the four strains of lactic acid bacteria were stored at 4°C with the extracted heme and living lactic acid bacterial cells were counted. There were more L. lactis and L. plantarum live cells when stored with heme, whereas L. rhamosus and L. casei showed no significant differences in live-cell numbers. The potential uses of the heme from C. glutamicum are further discussed.

Diffusible substances from lactic acid bacterial cultures exert strong inhibitory effects on Listeria monocytogenes and Salmonella enterica serovar enteritidis in a co-culture model

BackgroundFood-borne infections cause huge economic and human life losses. Listeria monocytogenes and Salmonella enterica serovar Enteritidis are among the top ranking pathogens causing such losses. Control of such infections is hampered by persistent contamination of foods and food-processing environments, resistance of pathogens to sanitizing agents, existence of heterogeneous populations of pathogens (including culturable and viable but non-culturable cells) within the same food items, and inability to detect all such pathogens by culture-based methods. Modern methods such as flow cytometry allow analyses of cells at the single cell level within a short time and enable better and faster detection of such pathogens and distinctions between live and dead cells. Such methods should be complemented by control strategies including the use of beneficial bacteria that produce metabolites capable of inhibiting food-borne pathogens. In this study, broth cultures of lactic acid bacteria (LAB) isolated from fermented milk were tested for production of substances capable of inhibiting L. monocytogenes and S. Enteritidis in co-culture with LAB by assessment of colony-forming units (CFU) and live:dead cell populations by flow cytometry.ResultsThe LAB isolates belonged to the species Lactococcus lactis, Enterococcus faecalis and Enterococcus faecium. Some LAB were effective in inhibition. Plating indicated up to 99% reduction in CFU from co-cultures compared to control cultures. Most of the bacteria in both cultures were in the viable but non-culturable state. The flow data showed that there were significantly higher dead cell numbers in co-cultures than in control cultures, indicating that such killing was caused by diffusible substances produced by the LAB cultures.ConclusionThis study showed that metabolites from selected local LAB species can be used to significantly reduce pathogen load. However, conditions of use and application need to be further investigated and optimized for large-scale utilization.