Batch fermentation of whey ultrafiltrate by Lactobacillus helveticus for lactic acid production

Summary Cheese whey ultrafiltrate (WU) was used as the carbon source for the production of lactic acid by batch fermentation with Lactobacillus helveticus strain milano. The fermentation was conducted in a 400 ml fermentor at an agitation rate of 200 rpm and under conditions of controlled temperature (42° C) and pH. In the whey ultrafiltrate-corn steep liquor (WU-CSL) medium, the optimal pH for fermentation was 5.9. Inoculum propagated in skim milk (SM) medium or in lactose synthetic (LS) medium resulted in the best performance in fermentation (in terms of growth, lactic acid production, lactic acid yield and maximum productivity of lactic acid), as compared to that propagated in glucose synthetic (GS) medium. The yeast extract ultrafiltrate (YEU) used as the nitrogen/growth factor source in the WU medium at 1.5% (w/v) gave the highest maximum productivity of lactic acid of 2.70 g/l-h, as compared to the CSL and the tryptone ultrafiltrate (TU). L. helveticus is more advantageous than Streptococcus thermophilus and Lactobacillus delbrueckii for the production of lactic acid from WU. The L. helveticus process will provide an alternative solution to the phage contamination in dairy industries using Lactobacillus bulgaricus.     

Optimization of lactic acid production from beet molasses by Lactobacillus delbrueckii NCIMB 8130

Production of lactic acid from beet molasses by Lactobacillus delbrueckii NCIMB 8130 in static and shake flask fermentation was investigated. Shake flasks proved to be a better fermentation system for this purpose. Substitution of yeast extract with other low cost protein sources did not improve lactic acid production. The maximum lactic acid concentration was achieved without treatment of molasses. A Central Composite Design was employed to determine the maximum lactic acid concentration at optimum values for the process variables (sucrose, yeast extract, CaCO₃). A satisfactory fit of the model was realized. Lactic acid production was significantly affected both by sucrose–yeast extract and sucrose–CaCO₃ interactions, as well as by the negative quadratic effects of these variables. Sucrose and yeast extract had a linear effect on lactic acid production while the CaCO₃ had no significant linear effect. The maximum lactic acid concentration (88.0 g/l) was obtained at concentrations for sucrose, yeast extract and CaCO₃ of 89.93, 45.71 and 59.95 g/l, respectively.     

Optimization of L(+)-lactic acid production employing statistical experimental design

Summary An orthogonal 2³-factorial experimental design was used to optimize L(+)-lactic acid production byLactobacillus casei. With a 22 % (v/v) inoculum the optimum concentration of yeast extract for maximum lactic acid concentration and yield was about 2 % (w/v) and that of the initial glucose 9 to 11 %.     

Genome shuffling of Lactobacillus delbrueckii mutant and Bacillus amyloliquefaciens through protoplasmic fusion for L-lactic acid production from starchy wastes

Current study was focused on the development of a non-fastidious lactic acid producing strain having better growth rate, low pH tolerance and good productivity by genome shuffling of a mutant strain of Lactobacillus delbrueckii NCIM 2025 and an amylase producing non-fastidious Bacillus amyloliquefaciens ATCC 23842. After the third cycle of the protoplast fusion, lactic acid production by few fusants was monitored and the best fusant was selected for further studies. Optimization of the important process parameters for lactic acid production was conducted using Plackett-Burman design and response surface methodology. Selected fusant could utilize the liquefied cassava bagasse starch directly with minimum nutrient supplementation for lactic acid production. During validation, 40g/L of lactic acid was obtained ( approximately 96% conversion of starch to lactic acid) by using fusant inoculum (3%, v/v) from 83g/L cassava bagasse (starch content 50% w/w) supplemented with yeast extract and peptone (0.2% each, w/v) and the buffering agent (2% CaCO(3), w/v).     

Production of propionic acid by mixed cultures ofPropionibacterium shermanii andLactobacillus casei in autoclave-sterilized whey

Summary Pure cultures ofPropionibacterium freudenreichii ss.shermanii did not grow in autoclave-sterilized cheese whey (121°C, 15 psi, 20 min) at whey concentrations greater than 2% (w/v) spray-dried sweet dairy whey. Propionic acid was produced from autoclave-sterilized whey by growingP. shermanii in mixed culture withLactobacillus casei. In medium containing 5–12% autoclaved whey solids and 1% yeast extract, the mixed culture produced 1.3–3.0% propionic acid, 0.5–1.0% acetic acid, and 0.05–0.80% lactic acid. All the lactose was consumed. Using pH-controlled fermentors (pH=7.0), mixed cultures produced at least 30% more propionic acid than cultures in which pH was not controlled.     

Solid-state fermentation for gluconic acid production from sugarcane molasses by Aspergillus niger ARNU-4 employing tea waste as the novel solid support

Solid-state fermentation (SSF) was evaluated to produce gluconic acid by metal resistant Aspergillus niger (ARNU-4) strain using tea waste as solid support and with molasses based fermentation medium. Various crucial parameters such as moisture content, temperature, aeration and inoculum size were derived; 70% moisture level, 30 degrees C temperature, 3% inoculum size and an aeration volume of 2.5l min(-1) was suited for maximal (76.3 gl(-1)) gluconic acid production. Non-clarified molasses based fermentation media was utilized by strain ARNU-4 and maximum gluconic acid production was observed following 8-12 days of fermentation cycle. Different concentrations of additives viz. oil cake, soya oil, jaggary, yeast extract, cheese whey and mustard oil were supplemented for further enhancement of the production ability of microorganism. Addition of yeast extract (0.5%) was observed inducive for enhanced (82.2 gl(-1)) gluconic acid production.     

Effect of carbon and nitrogen sources on growth and biological efficacy of Pseudomonas fluorescens and Bacillus subtilis against Rhizoctonia solani, the causal agent of bean damping-off

One of the most important environmental factors that regulate the growth and antagonistic efficacy of biocontrol agents is the medium. The aim of this paper was to find the nitrogen and carbon sources that provide maximum biomass production of strains P-5 and P-6 (Pseudomonas fluorescens), B-3 and B-16 (Bacillus subtilis) and minimum cost of media, whilst maintaining biocontrol efficacy. All of the strains were grown in seven liquid media (pH=6.9) including: sucrose + yeast extract, molasses of sugar beet + yeast extract in 2:1 and 1:1 w/w ratios, molasses of sugar beet + urea, nutrient broth, molasses and malt extract, at an initial inoculation of 1 x 10(5) CFU ml(-1). Cells from over night cultures used to inoculate soil at 1 x 10(9) CFU cm(-3) soil. At the same time, fungal inoculum (infected millet seed with Rhizoctonia solani) was added to soil at the rate of 2 g kg(-1) soil. Results indicated that growth of P-6, B-3 and B-16 in molasses + yeast extract (1:1 w/w) medium was significantly higher than in the other media. Molasses + yeast extract (1:1 and 2:1 w/w) media supported rapid growth and high cell yields in P-5. In greenhouse condition, results indicated that the influence of the media on the biocontrol efficacy of P-5, P-6, B-3 and B-16 was the same and Pseudomonas fluorescens P-5 in molasses and malt extract media reduced the severity of disease up to 72.8 percent. On the other hand, there were observed significant differences on bean growth after one month in greenhouse. P-5 in molasses + yeast extract (1:1 w/w) medium had the most effects on bean growth promotion. In this study molasses media showed good yield efficacy in all of the strains. The high sucrose concentration in molasses justifies the high biomass in all of the strains. Also, the low cost of molasses allows its concentration to be increased in media. On the other hand, yeast extract was the best organic nitrogen source for antagonist bacteria but it is expensive for an industrial process. So it should be replaced by another industrial product instead of yeast extract, which confirm by an economic and technological study. The results obtained in this study could be used to provide a reliable basis to increase the population of biocontrol agents in fermentation process.     

Production of ethanol from the mixture of beet molasses and cheese whey by a 2-deoxyglucose-resistant mutant of Kluyveromyces marxianus

Fourteen lactose-fermenting strains of Kluyveromyces marxianus , including its anamorph, Candida kefyr , were grown in two media containing 20% (w/v) sugar as either beet molasses or cheese whey. Strain NBRC 1963 of K. marxianus converted sucrose and lactose to ethanol in both media most efficiently. However, ethanol was produced from sucrose and not from lactose by strain NBRC 1963 in the medium containing equal amounts of sugar from beet molasses and cheese whey. The spontaneous mutants resistant to 2-deoxyglucose in the minimal medium composed of galactose as the sole carbon source were isolated from strain NBRC 1963. Among them, strain KD-15 vigorously produced ethanol in the media containing beet molasses, cheese whey, or both. The mutant strain KD-15 was insensitive to catabolite repression, as shown by the observation that β-galactosidase was not repressed in the presence of sucrose from beet molasses.     

Cauliflower waste incorporation into cane molasses improves ethanol production using <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> MTCC 178

Diluted cane molasses having total sugar and reducing sugar content of 9.60 and 3.80% (w/v) respectively was subjected to ethanol production by Saccharomyces cerevisiae MTCC 178. Incorporation of dried Cauliflower Waste (CW) in molasses at the level of 15 % increased ethanol production by nearly 36 % compared to molasses alone. Addition of 0.2 % yeast extract improved ethanol production by nearly 49 % as compared to molasses alone. When the medium containing diluted molasses and 0.2 % yeast extract was supplemented with 15 % CW, 29 % more ethanol was produced compared to molasses with 0.2 % yeast extract. Cell biomass, ethanol production, final ethanol concentration and fermentation efficiency of 2.65 mg mL⁻¹, 41.2 gL⁻¹, 0.358 gg⁻¹ and 70.11 % respectively were found to be best at 15% CW supplementation level besides reduction in fermentation time but further increase in CW level resulted in decline on account of all the above parameters. This is probably the first report to our knowledge, in which CW was used in enhancing ethanol production significantly using a small quantity of yeast extract.     

Production of <Emphasis Type="SmallCaps">d</Emphasis>-lactic acid from sugarcane molasses,sugarcane juice and sugar beet juice by <Emphasis Type="Italic">Lactobacillus delbrueckii</Emphasis>

Lactobacillus delbrueckii was grown on sugarcane molasses, sugarcane juice and sugar beet juice in batch fermentation at pH 6 and at 40°C. After 72 h, the lactic acid from 13% (w/v) sugarcane molasses (119 g total sugar l⁻¹) and sugarcane juice (133 g total sugar l⁻¹) was 107 g l⁻¹ and 120 g l⁻¹, respectively. With 10% (w/v) sugar beet juice (105 g total sugar l⁻¹), 84 g lactic acid l⁻¹ was produced. The optical purities of d-lactic acid from the feedstocks ranged from 97.2 to 98.3%.     

Physiological restriction of the L-lactic acid production by Rhizopus arrhizus

The influence of basic physiological factors on the quality of inocula and L(+)-lactic acid production by Rhizopus arrhizus CCM 81 09 were studied. The most effective preparation of the spores (5 × 10⁷ spores/ml) and subsequent good lactate production was achieved on the agar medium with soil extract and malt agar. The optimum initial amount of active spores for inoculation was 10³–10⁴ spores/ml. The preparation of inoculum required intensive stirring with lower aeration and pH maintained in the range from 4.8 to 6.0 by the addition of CaCO₃. The maximum yield of lactic acid production was achieved by using 5% (v/v) of 24-h-old inoculum. The intensity of lactic acid production in the inoculum was proportional to its production in the subsequent steps of fermentation and can be used as a fast control of the physiological state of the producers.     

Optimization of pH,Temperature and Inoculum Ratio for the Production of β‐D‐Galactosidase by Kluyveromyces marxianus Using a Lactose‐free Medium

The pH, temperature and inoculum ratio for the production of β‐galactosidase by Kluyveromyces marxianus CDB 002 were optimized using sugar‐cane molasses (100 g/l) in a lactose‐free medium. The temperature optimum was evaluated in the range from 28–37 °C. Lactase production was initiated after substrate consumption indicating a reversible enzyme inhibition or catabolic repression. The specific enzyme activity after 45 h was between 456.3 U/g cell mass (37 °C) and 733.3 U/g (34 °C), whereas the highest volumetric activity was obtained at 30 °C: 21.8 U/ml. This is generally consistent with results from other authors that used whey as a carbon source. Ethanol as a by‐product reached its maximum concentration after 10–14 h (31.1–40.5 g/l), but was completely consumed afterwards. A pH of 5.5 without further control gave the best production rate for lactase (484.4 U/l × h). In this process, the pH was stable during cell growth at 5.5 and then went up to pH 7.2 after 45 h. At a fixed pH of 5.5 or 6.5, the production rates achieved 313.3 U/l × h and 233.3 U/ l × h, respectively. These results differed from those of other authors, who suggested a fixed pH at 7.0 using whey as a carbon source. There were no significant differences between inoculum ratios of 1% [v/v] and 10% [v/v] so that 1% is the preferable ratio as it is cheaper. Yeast extract (10 g/l) and peptone (20 g/l) were used as the vitamin and nitrogen source, respectively, for the studies of temperature and pH. These were substituted by corn steep liquor (100 g/l) for inoculum ratio experiments. Production of lactase using sugar cane molasses in a lactose‐free medium gave better enzyme productivity rates than obtained by other authors using whey. The optimum conditions for β‐galactosidase synthesis were a temperature of 30–34 °C and an inoculum ratio of 1% [v/v], an initial pH of 5.5 without any further control or a control of 5.5 during cell growth. Then the pH was raised up to 7.     

Sugarcane molasses and yeast powder used in the Fructooligosaccharides production by Aspergillus japonicus-FCL 119T and Aspergillus niger ATCC 20611

Different concentrations of sucrose (3–25% w/v) and peptone (2–5% w/v) were studied in the formulation of media during the cultivation of Aspergillus japonicus-FCL 119T and Aspergillus niger ATCC 20611. Moreover, cane molasses (3.5–17.5% w/v total sugar) and yeast powder (1.5–5% w/v) were used as alternative nutrients for both strains’ cultivation. These media were formulated for analysis of cellular growth, β-Fructosyltransferase and Fructooligosaccharides (FOS) production. Transfructosylating activity (U _t ) and FOS production were analyzed by HPLC. The highest enzyme production by both the strains was 3% (w/v) sucrose and 3% (w/v) peptone, or 3.5% (w/v) total sugars present in cane molasses and 1.5% (w/v) yeast powder. Cane molasses and yeast powder were as good as sucrose and peptone in the enzyme and FOS (around 60% w/w) production by studied strains.     

Production of alcohol from sugar beet molasses without heat or filter sterilization

Among three esters of p-hydroxybenzoate, n-butyl p-hydroxybenzoate was selected as the best antimicrobial substance. Molasses medium sterilized by this ester was used as a substrate for ethanol production. n-Butyl p-hydroxybenzoate (0.15% w/v) completely inhibited the growth of free yeast cell inoculum, Ca-alginate immobilized yeast inoculum and bacterial contaminants. Immobilization of the yeast cell inoculum in Ca-alginate with castor oil (6% v/v) offered a yeast cell protection against the inhibitory effect of n-butyl p-hydroxybenzoate. The presence of castor oil in this immobilization system did not affect the metabolic activity of the yeast in beads compared to the cells immobilized without castor oil. The yeast cell beads in this system completely utilized up to 25% molasses sugar with an ethanol yield of 10.58%, equal to 83% of its theoretical value. The beads were stable and could be used successfully for seven cycles of batch fermentation. The optimum fermentation temperature using this system was 35°C. Received 21 January 1997/ Accepted in revised form 05 May 1997     

Suitability of commercial beet molasses fractions as substrates fro polyhydroxyalkanoate production byAzotobacter vinelandii UWD

Summary Beet molasses that had been fractionated commercially by ion exclusion resulted in two waste-streams: extract molasses (EM) and concentrated separator by-product (CSB). Only EM at 4–5% w/v contained sufficient sugar to promote polyhydroxybutyrate (PHB) formation byAzotobacter vinelandii UWD, but the yield of PHB/protein was less than that obtained in unfractionated beet molasses. EM and especially CSB added at 0.5–2.0% w/v to media containing a variety of sugar sources promoted an increased yield of PHB/protein. The best use of these beet molasses fractions was, therefore, as a minor addition to media containing sugars to increase PHB yield, but not as a primary substrate for PHB production.     

Production of Bakers' Yeast in Cheese Whey Ultrafiltrate

A process for the production of bakers' yeast in whey ultrafiltrate (WU) is described. Lactose in WU was converted to lactic acid and galactose by fermentation. Streptococcus thermophilus was selected for this purpose. Preculturing of S. thermophilus in skim milk considerably reduced its lag. Lactic fermentation in 2.3×-concentrated WU was delayed compared with that in unconcentrated whey, and fermentation could not be completed within 60 h. The growth rate of bakers' yeast in fermented WU differed among strains. The rate of galactose utilization was similar for all strains, but differences in lactic acid utilization occurred. Optimal pH ranges for galactose and lactic acid utilization were 5.5 to 6.0 and 5.0 to 5.5, respectively. The addition of 4 g of corn steep liquor per liter to fermented WU increased cell yields. Two sources of nitrogen were available for growth of Saccharomyces cerevisiae: amino acids (corn steep liquor) and ammonium (added during the lactic acid fermentation). Ammonium was mostly assimilated during growth on lactic acid. This process could permit the substitution of molasses by WU for the industrial production of bakers' yeast.     

Use of mixed cultures for the fermentation of cereal-based substrates with potential probiotic properties

The production of a new cereal-based probiotic foods with suitable aroma, flavor and pH using mixed culture fermentation has been investigated. This required the selection of suitable types of cereal grains and probiotic microorganisms. In a medium of 5% (w/v) malt suspension the effects of yeast presence on the fermentation of a lactic acid bacterium (LAB), Lactobacillus reuteri, was studied. With different inoculum ratios between the yeast and the LAB, the characteristics of the fermentation broth including pH and the contents of free amino nitrogen (FAN), reducing sugar, lactic acid and ethanol were investigated. It was found that LAB growth was enhanced by the introduction of the yeast. In mixed culture broth pH was lowered and the production of lactic acid and ethanol were increased in comparison against pure LAB culture.     

Molasses and microbial inoculants improve fermentability and silage quality of cotton waste-based spent mushroom substrate

A small-silo study was conducted to develop an effective ensiling storage method for the use of cotton waste-based spent mushroom substrate (SMS) as an animal feed. The SMS was ensiled with 5% molasses (DM basis), 0.5% (v/w) lactic acid bacteria (LAB, Lactobacillus plantarum) inoculant or 0.5% (v/w) yeast (Saccharomyces cerevisiae) inoculant. The treatments included 100% SMS (control), 95% SMS+5% molasses (T1), 95% SMS+5% molasses+0.5% LAB (T2) and 95% SMS+5% molasses+5% LAB+0.5% yeast (T3). The treatments were ensiled for 10. Change in chemical compositions was little (P>0.05) according to the ensiling process and treatments. Compared with those before ensiling, 100% SMS (control) after ensiling showed unstable fermentative properties with high pH (5.2) and little lactic acid production. Compared with the ensiled control, treatments (T1, T2 and T3) resulted in decreased pH, 18-20 times higher concentrations of lactic acid, and greater populations of total bacteria (P<0.07), LAB and yeast (P<0.07). The addition of 5% molasses, 0.5% LAB and 0.5% yeast (T3) to the SMS resulted in the lowest pH (4.25) and the greatest microbial populations. Treatment T3 was selected for a large scale silo study which was ensiled for 10, 20 and 30 d. As in the small-silo study, the T3 treatment showed favorable fermentative and microbial parameters, compared with the control, by decreasing pH and increasing lactic acid concentrations, LAB and yeast populations. The minimum ensiling period was 20 d, when pH was reasonably low and LAB and yeast populations were greatest. In conclusion, molasses and microbial inoculation improved silage quality of SMS.     

Effect of pH and lactic or acetic acid on ethanol productivity by Saccharomyces cerevisiae in corn mash

The effects of lactic and acetic acids on ethanol production by Saccharomyces cerevisiae in corn mash, as influenced by pH and dissolved solids concentration, were examined. The lactic and acetic acid concentrations utilized were 0, 0.5, 1.0, 2.0, 3.0 and 4.0% w/v, and 0, 0.1, 0.2, 0.4, 0.8 and 1.6% w/v, respectively. Corn mashes (20, 25 and 30% dry solids) were adjusted to the following pH levels after lactic or acetic acid addition: 4.0, 4.5, 5.0 or 5.5 prior to yeast inoculation. Lactic acid did not completely inhibit ethanol production by the yeast. However, lactic acid at 4% w/v decreased (P<0.05) final ethanol concentration in all mashes at all pH levels. In 30% solids mash set at pH ≤5, lactic acid at 3% w/v reduced (P<0.05) ethanol production. In contrast, inhibition by acetic acid increased as the concentration of solids in the mash increased and the pH of the medium declined. Ethanol production was completely inhibited in all mashes set at pH 4 in the presence of acetic acid at concentrations ≥0.8% w/v. In 30% solids mash set at pH 4, final ethanol levels decreased (P<0.01) with only 0.1% w/v acetic acid. These results suggest that the inhibitory effects of lactic acid and acetic acid on ethanol production in corn mash fermentation when set at a pH of 5.0–5.5 are not as great as that reported thus far using laboratory media.     

Production of Bacteriocin ST33LD, Produced by Leuconostoc mesenteroides subsp. mesenteroides, as Recorded in the Presence of Different Medium Components

Summary Bacteriocin ST33LD, produced by Leuconostoc mesenteroides subsp. mesenteroides, is approximately 2.7 kDa in size and inhibits Enterococcus faecalis, Escherichia coli, Lactobacillus casei and Pseudomonas aeruginosa. Good growth was recorded in the presence of 10% (w/v) soy milk or 10% (w/v) molasses, but there was no bacteriocin production. Growth in MRS broth adjusted to pH 4.5 yielded low bacteriocin levels (800 AU/ml). However, the same medium adjusted to pH 5.0, 5.5 and 6.5, respectively, yielded 3200 AU/ml. Tween 80 decreased bacteriocin production by more than 50%. Growth in the presence of tryptone yielded maximal activity (12,800 AU/ml), whereas different combinations of tryptone, meat extract and yeast extract produced activity levels of 1600 AU/ml and less. Growth in the presence of 2.0% (w/v) sucrose, or maltose, yielded much higher levels of bacteriocin activity (12,800 AU/ml) compared to growth in the presence of 2.0% (w/v) glucose or lactose (6400 AU/ml). Lower yields were also recorded in the presence of fructose and mannose. KH₂PO₄ at 10.0% (w/v) stimulated bacteriocin production. Glycerol concentrations of 0.5% (w/v) and higher (up to 5.0%, w/v) repressed bacteriocin production by 50%. The addition of cyanocobalamin, thiamine and L-ascorbic acid to MRS broth (1.0 ppm) yielded 12,800 AU/ml bacteriocin, whereas the addition of DL-6,8-thioctic acid yielded only 6 400 AU/ml.