Influence of growth supplements on lactic acid production in whey ultrafiltrate by Lactobacillus helveticus

Summary Investigations have been carried out on lactic acid production by Lactobacillus helveticus CNRZ 303 in whey ultrafiltrate. Addition of beet molasses was investigated with good results, although yeast extract proved to be more effective. The size of inoculum and the preculture medium also played a significant role in determining the amount of lactic acid produced during the fermentation process. High lactose consumption (94.09%), together with good lactic acid production (26.09 g/l) and yield (0.90%), were obtained in whey ultrafiltrate supplemented with 1% (w/v) beet molasses (WUM), with a 10% (w/v) inoculum and peptonized milk as preculture medium. Although these results were similar to those obtained when yeast extract was used as supplement, the maximum volumetric productivities proved to be quite different, and were definitely higher with yeast extract.Offprint requests to: L. Chiarini     

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.     

The production of lactic acid from whey permeate byLactobacillus helveticus

Summary The effect of pH on growth and lactic acid production ofLactobacillus helveticus was investigated in a continuous culture using supplemented whey ultrafiltrate. Maximum lactate productivity of 5 gl^–1h^–1 occurred at pH 5.5. Whey permeates concentrated up to four times were fermented using batch cultures. Maximum lactic acid concentration of 95 gl^–1 was attained, but residual sugars indicated a possible limitation in growth factors.Nomenclature D Dilution rate [h^–1] - X Biomass [gl^–1] - Glu Glucose consentration [gl^–1] - Gal Galactose consentration [gl^–1] - S Substrate, Lactose consentration [gl^–1] - P Product, Lactate consentration [gl^–1] - Y_p/s Yield, defined as P/S [gg^–1] - r_i Rate of synthesis or consumption of i [gl^–1h^–1]     

Batch propagation of Lactobacillus helveticus for production of lactic acid from lactose concentrated cheese whey with microaeration and nutrient supplementation

Continuous mix batch bioreactors were used to study the kinetic parameters of lactic acid fermentation in microaerated-nutrient supplemented, lactose concentrated cheese whey using Lactobacillus helveticus. Four initial lactose concentrations ranging from 50 to 150 g l^–1 were first used with no microaeration and no yeast extract added to establish the substrate concentration above which inhibition will occur and then the effects of microaeration and yeast extract on the process kinetic parameters were investigated. The experiments were conducted under controlled pH (5.5) and temperature (42 °C) conditions. The results indicated that higher concentrations of lactose had an inhibitory effect as they increased the lag period and the fermentation time; and decreased the specific growth rate, the maximum cell number, the lactose utilization rate, and the lactic acid production rate. The maximum lactic acid conversion efficiency (75.8%) was achieved with the 75 g l^–1 initial lactose concentration. The optimum lactose concentration for lactic acid production was 75 g l^–1 although Lactobacillus helveticus appeared to tolerate up to 100 g l^–1 lactose concentration. Since the lactic acid productivity is of a minor importance compared to lactic acid concentration when considering the economic feasibility of lactic acid production from cheese whey using Lactobacillus helveticus, a lactose concentration of up to 100 g l^–1 is recommended. Using yeast extract and/or microaeration increased the cell number, specific growth rate, cell yield, lactose consumption, lactic acid utilization rate, lactic acid concentration and lactic acid yield; and reduced the lag period, fermentation time and residual lactose. Combined yeast extract and microaeration produced better results than each one alone. From the results it appears that the energy uncoupling of anabolism and catabolism is the major bottleneck of the process. Besides lactic acid production, lactose may also be hydrolysed into glucose and galactose. The -galactosidase activity in the medium is caused by cell lysis during the exponential growth phase. The metabolic activities of Lactobacillus helveticus in the presence of these three sugars need further investigation.     

Uptake of lactose and continuous lactic acid fermentation by entrapped non-growing Lactobacillus helveticus in whey permeate

 Continuous production of lactic acid from lactose has been carried out in a stirred-tank reactor with non-growing Lactobacillus helveticus entrapped in calcium alginate beads. A considerably longer operation half-life was obtained in a continuously operated reactor than in a batch-operated reactor. It is possible to simulate the action of entrapped non-growing cells on the basis of information from diffusion and kinetic experiments with suspended free cells. The simulation fit the experimental data over a broad range of substrate concentrations if the specific lactic acid production rate, q _P, was used as a variable parameter in the model. The dynamic mathematical model used is divided into three parts: the reactor model, which describes the mass balance in a continuously operated stirred-tank reactor with immobilized biomass, the mass-transfer model including both external diffusion and internal mass transfer, and the kinetic model for uptake of substrate on the basis of a Michaelis-Menten-type mechanism. From kinetic data obtained for free biomass experiments it was found, with the use of non-linear parameter estimation techniques, that the conversion rate of lactose by L. helveticus followed a Michaelis-Menten-type mechanism with K _S at half-saturation=0.22±0.01 g/l. The maximum specific lactose uptake rate for growing cells, q _S,max, varied between 4.32±0.02 g lactose g cells^-1 h^-1 and 4.89 ±0.02 g lactose g cells^-1 h^-1. The initial specific lactose uptake rate for non-growing cells, q _S,0, was found to be approximately 40% of the maximum specific lactose uptake rate for growing cells. Received: 4 October 1995/Received last revision: 23 April 1996/Accepted: 29 April 1996     

Efficiency of lactic acid production by Lactobacillus helveticus in a membrane cell recycle reactor

Abstract: An economic evaluation is presented of lactic acid production in a membrane cell recycle reactor. From this evaluation it is concluded that the economic feasibility of the process is primarily limited by production capacity and product concentration and to a lesser extent by productivity. In membrane cell recycle reactor experiments and batch cultivation experiments with Lactobacillus helreticus , it is shown that the economic feasibility of the process using this organism is limited by organic acid inhibition resulting in energy uncoupling of anabolism and catabolism. Due to this inhibition, the maximum lactic acid concentration that can be obtained in the membrane reactor process is 50 g I^1—. Furthermore it is shown that not only the fermentative conversion of lactose into lactic acid but also the hydrolysis of lactose into glucose and galactose is an important process. The β-galaetosidase activity needed for the hydrolysis is generated during the exponential growth phase of Lb. helveticus     

Lactic acid production from whey permeate by immobilized Lactobacillus casei

Two matrices have been assessed for their ability to immobilize Lactobacillus casei cells for lactic acid fermentation in whey permeate medium. Agar at 2% concentration was found to be a better gel than polyacrylamide in its effectiveness to entrap the bacterial cells to carry out batch fermentation up to three repeat runs. Of the various physiological parameters studied, temperature and pH were observed to have no significant influence on the fermentation ability of the immobilized organism. A temperature range of 40–50°C and a pH range of 4.5–6.0 rather than specific values, were found to be optimum when fermentation was carried out under stationary conditions. In batch fermentation ～90% conversion of the substrate (lactose) was achieved in 48 h using immobilized cell gel cubes of 4 × 2 × 2 mm size, containing 400 mg dry bacterial cells per flask and 4.5% w/v (initial) whey lactose content as substrate. However, further increase in substrate levels tested (>4.5% w/v) did not improve the process efficiency. Supplementation of Mg²⁺ (1 mM) and agricultural by-products (mustard oil cake, 6%) in the whey permeate medium further improved the acid production ability of the immobilized cells under study.     

Seed culture and its effect on the growth and lactic acid production of Lactobacillus helveticus

Effects of seed culture medium on the subsequent culture of L. helveticus growing on whey supplemented with yeast extract and peptones have been examined. Cells were inoculated when the maximum cellular activity was achieved, i.e. the maximum for the target function, the product of the specific growth rate with the undissociated lactic acid concentration. This function decreased when the nitrogen supplementation of the preculture medium was lowered, resulting only in an increase of the lag phase length of the culture, corresponding to a cellular adaptation to the new medium. On the contrary, when cells were not in the same physiological state, growth and production parameters were affected, maximum rates, as well as maximum biomass concentration. This occurred in case of the comparison of inocula carried out under pH control or not, or when various preculture lengths were tested.     

A continuous lactic acid production system using an immobilized packed bed of Lactobacillus helveticus

A 5 l packed bed bioreactor was used to study the effect of initial lactose concentration and hydraulic retention time (HRT) on cell growth, lactose utilization and lactic acid production. Up to 95% of the initial lactose concentration was utilized at longer HRTs (30-36 h). The study showed that lactic acid production increased with increases in HRT (12-36 h) and initial lactose concentrations. The highest lactic acid production rate (3.90 g l(-1) h(-1)) was obtained with an initial lactose concentration of 100 g/l and an HRT of 18 h, whereas the lowest lactic acid production rate (1.35 g l(-1) h(-1)) was obtained with an initial lactose concentration of 50 g/l and an HRT of 36 h. This suggested that optimal lactic acid production can be achieved at an HRT of 18 h and initial lactose concentration of 100 g/l.     

Batch cultures of supplemented whey permeate using Lactobacillus helveticus: unstructured model for biomass formation, substrate consumption and lactic acid production

The Luedeking and Piret expression can not account for the cessation of production observed at the end of batch; so an empiric term has been previously added to this equation which accounted in a global way for possible substrate limitations. In the model developed in this work, a carbon substrate limitation appeared explicitly in the production expression. Assuming a sigmoidal variation with time of specific growth rate previously validated, the new production model matched well the entire experimental production kinetics. It has been successfully tested for a wide range of nitrogen supplementations, i.e. from an almost total coupling between growth and production for largely supplemented media, to a high decoupling in case of few available nitrogen. Since all the parameters of this model have an obvious biologic meaning, it may be an unvaluable tool for the comprehension of the phenomenon. The model accounted also well for the variation of the specific production rate versus specific growth rate, avoiding the noise due to the direct differentiation of experimental data.     

Limitation of growth and lactic acid production in batch and continuous cultures of Lactobacillus helveticus

Summary Continuous and batch cultures of Lactobacillus helveticus operated under different conditions were studied with respect to the limitation of growth and lactic acid production by increasing undissociated lactic acid and hydrogen ion concentrations, respectively. In a single-stage continuous culture without pH control a final pH of 3.8 and 65 mm undissociated lactic acid was obtained. In two-stage continuous cultures provided with different growth media and run at different pH values, 65–70 mm free acid was obtained in the second stage. Further batch-culture experiments showed growth limitation at 60–70 mm lactic acid. After growth ceased, production of lactate continued until a lactic acid concentration of about 100 mm was reached; obviously an uncoupling of growth and acid production had occurred. Examining the effect of different concentrations of either lactic acid or hydrochloric acid, added to growing batch cultures of L. helveticus, it was shown that the undissociated lactic acid concentration was responsible for growth limitation and lactic acid production in this organism, whereas the pH value had only an indirect effect.     

Behaviour of different Lactobacillus species in the production of lactic acid by the fermentation of whey

The behaviour of different Lactobacillus casei and Lactobacillus plantarum species in the fermentation of Manchego whey was experimentally studied and the results were statistically analyzed using a hypothesis contrast method. The steadiness of the velocity of the production of lactic acid during the fermentation process allowed the use of this variable to compare the different microorganisms. From this comparison it was inferred that the individuals of the same population behave alike and that the L. casei population produces lactic acid at a higher rate than the L. plantarum population. A competitive effect among the members of the L. casei population was also observed.     

Semi-continuous lactic fermentation of whey by Lactobacillus bulgaricus

Semi-continuous tests of lactic fermentation of whey by Lactobacillus buigaricus were carried out using a mixture of hydrolysed milk and vitamins. The volume of the Inoculum varied from 20% to 50% of the reactor working volume. A Monod-like equation correlates the lactic acid productivity and the volume fraction of inoculum.The authors are with the Centro de Desenvolvimento Biotecnológico de Santa Catarina (Biotechnological Development Centre of Santa Catarina), R. Princesa isabel, 114, 89200, Joinville, SC, Brazil. CDEB. L.A. Kulay was an undergraduate student, in 1989, of the Escola de Engenharia Mauá (Mauá Engineering School), São Caetano do Sul, SP, Brazil. W. Borzani is the corresponding author.     

Lactic acid production rates during the different growth phases of Lactobacillus helveticus cultivated on whey supplemented with yeast extract

The influence of the yeast extract concentration on the kinetic relationship between growth and production has been analyzed in detail by comparing growth and production rates instead of specific rates. The highest average acid production rate occurred during the slowing down phase of growth. Irrespective of the nitrogen supplementation, the contribution to the total acid production of this phase was nearly the same as that of the growth-associated production one. © Rapid Science Ltd. 1998     

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.     

Analysis of promoter sequences from Lactobacillus helveticus CNRZ32 and their activity in other lactic acid bacteria

AIMS: To clone and analyse seven putative promoter fragments (pepC, pepN, pepX, pepO, pepE, pepO2, hsp17) from Lactobacillus helveticus CNRZ32 for their expression in Lact. helveticus CNRZ32, Lact. casei ATCC334 and Lactococcus lactis MG1363. METHODS AND RESULTS: Promoter fragments were fused to the promoter-less beta-glucuronidase (gusA) gene on pNZ272(RBS-) (ATG-). The resulting constructs were evaluated for their ability to drive the expression of active GusA with 0.5 mmol l(-1) 5-bromo-4-chloro-3-indolyl-beta-D-glucuronide. All promoters except P(pepN)::gusA were active in the examined strains. Northern hybridization was performed to examine the promoter strength. Sequence analysis of these promoters identified well conserved putative ribosomal binding and putative -10 hexamers sites. CONCLUSIONS: Seven promoter fragments from Lact. helveticus CNRZ32 were recognized in the lactic acid bacteria, Lact. casei ATCC334 and L. lactis MG1363, as well as in Escherichia coli. P(pepN)::gusA could not be maintained in the strains examined because of toxicity associated with heterologous protein over-expression driven by P(pepN). SIGNIFICANCE AND IMPACT OF THE STUDY: This study revealed that desirable levels of heterologous food-grade protein production in GRAS organisms can be obtained with the application of natural promoter fragments from closely related organisms.     

Influence of the metabolism pathway on lactic acid production from hemicellulosic trimming vine shoots hydrolyzates using Lactobacillus pentosus

Hemicellulosic hydrolyzates from trimming wastes of vine shoots were proposed as a carbon source for lactic acid production by Lactobacillus pentosus CECT-4023T (ATCC-8041). These hydrolyzates are composed mainly of glucose (12.0 g/L), xylose (17.5 g/L) and arabinose (4.3 g/L). Acetic acid, the main subproduct, started to be produced after all of the glucose was completely depleted, showing that the acetic acid coproduction came only from the xylose and arabinose consumption. In the absence of glucose, the L. pentosus pathway shifts from homo to heterofermentative. Thus, L. pentosus can be considered a facultative heterofermentative organism, degrading hexoses (glucose) via the Embden-Meyerhoff-Parnas pathway and pentoses (xylose and arabinose) via the phosphoketolase pathway. Hydrolyzates were vacuum evaporated to increase the initial sugars concentration up to 35.4 g/L of glucose, 52.3 g/L of xylose, and 13.0 g/L of arabinose. Under these conditions the lactic acid concentration reached 46.0 g/L (Q(P) = 0.933 g/L.h, Y(P/S) = 0.78 g/g; Y(P/S) theoretical = 91.7%) and a clear product inhibition was observed. Additional experiments with synthetic sugars, in the absence of inhibitory compounds, indicate that this inhibition must be attributed to the metabolic pathway but not to the inhibitory compounds present in the fermentation broth.     

利用氧化还原电位调控乳酸发酵

研究了控制不同氧化还原电位（oxidation—reduction potential,ORP）对乳酸发酵过程的影响。通过5L发酵罐分批发酵实验发现ORP水平控制在-170mV时最有利于乳酸生成,乳酸最高质量浓度达176g／L,糖酸转化率为94％,其平均乳酸产率3．7g/（L·h）,比ORP控制在-220mV和-120mV时分别高19％,37％;通过对发酵过程胞外有机酸浓度及代谢流分析发现氧化还原电位是通过影响细胞内代谢流分布来影响乳酸合成的。     

Effects of sodium acetate on the production of stereoisomers of lactic acid by Lactobacillus sakei and other lactic acid bacteria

Lactobacillus sakei and other lactic acid bacteria were studied on the change of the type of stereoisomers (the ratio of L-form to D-form) of lactic acid produced in the presence of sodium acetate and under other cultural conditions. Of 49 strains tested, only L. sakei NRIC 1071(T) and L. coryniformis subsp. coryniformis NRIC 1638(T) changed the type in the presence of 50 mm sodium acetate compared with the absence of sodium acetate. The type produced by L. sakei NRIC 1071(T) was shifted 30% or more from the DL-type to the L-type in the presence of 50 mm sodium acetate. L. sakei NRIC 1071(T) produced not only twice or more the amount of L-lactic acid but decreased the amount of D-lactic acid compared with the absence of sodium acetate. The shift of the DL-type to the L-type by L. sakei is due to the high production of L-lactic acid and the low production of D-lactic acid. The type of stereoisomers produced by 11 L. sakei strains was also shifted from the DL-type to the L-type in the presence of 50 mm sodium acetate. The shift of stereoisomers by the majority of L. sakei strains seems interesting from the viewpoint of the delineation of this species.