Continuous manufacture of yogurt. I. Optimal conditions and kinetics of the prefermentation process

The principles of a method for the continuous manufacture of yogurt, based upon a two stage system, are given. The first stage, the prefermentation of milk at 45°C to a pH of 5.7, is described. The limitations of this continuous prefermentation are experimentally determined. No change in the balance of the yogurt bacteria, Lactobacillus bulgaricus and Streptococcus thermophilus, was observed. A mathematical approach is given for starting and stopping the process.     

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

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

Analysis of pH-stat continuous cultivation and the stability of the mixed fermentation in continuous yogurt production

A pH-stat fermentor is a continuous cultivator in which the feed rate is controlled to maintain a constant pH, i.e., end-product acid concentration. This fermentor has application to the continuous cultivation of lactic acid-producing organisms in milk-based media. The equations describing the operation of this fermentor are developed. It is shown that, where the limiting substrate is the carbon and energy source, the operation of the fermentor is essentially equivalent to that of a turbidostat. In contrast to this, where the carbon and energy source is in excess and growth is limited by another substrate, pH-state fermentation is stable both in regions of substrate excess, where D = μ_max, comparable with turbidostat operation, and substrate limitation where D < μ_max, comparable with chemostat operation. These conditions are met in milk-based media. An analysis is presented, allowing the prediction of the degree of substrate limitation, cell density, and dilution rate in a pH-stat fermentor from batch-growth kinetics. This was confirmed using experimental data for the growth of Streptococcus thermophilus TS2 and Lactobacillus LB1 in skim milk. Stable simultaneous growth of two organisms in continuous culture occurs if their growth rates are determined by separate conditions, so that, at steady state, their growth rqtes are separately madeequal to the dilution rate. It is then predicted, and confirmed by experiment, that a mixed culture of S. thermophilus TS2 and L. bulgaricus LB1 in a pH-stat continuous fermentor in yogurt mix at pH 5.5 would be stable with the growth of L. bulgaricus being substrate unlimited and the fermentor operting with D = μ_max for L. bulgaricus LB1, and the growth of S. thermophilus TS2 being substrate limited so that its growth rate is equal to the existing dilution rate. Finally, it is predicted and confirmed by experiment that if the conditions are altered so that the growth of S. thermophilus TS2 is substrate unlimited the stable association is broken down, the fermentor operates with D approaching μ_max for S. thermophilus TS2, and L. bulgaricus LB1 is washed out to the level maintained by wall growth.     

Production and monomer composition of exopolysaccharides by yogurt starter cultures

As components of starter cultures for Bulgarian yogurt, Streptococcus salivarius subsp. thermophilus and Lactobacillus delbrueckii subsp. bulgaricus revealed extensive exopolysaccharide (EPS) production activity when cultivated in whole cow's milk. The polymer-forming activity of thermophilic streptococci was lower (230-270 mg EPS/L) than that of the lactobacilli (400-540 mg EPS/L). Mixed cultures stimulated EPS production in yogurt manufacture, and a maximum concentration of 720-860 mg EPS/L was recorded after full coagulation of milk. The monomer structure of the exopolysaccharides formed by the yogurt starter cultures principally consists of galactose and glucose (1:1), with small amounts of xylose, arabinose, and/or mannose.     

A continuous,multistage tower fermentor. II. Analysis of reactor performance

A method for analyzing the reactor behavior of a continuous, multistage tower fermentor is described. A model consisting of a system of interconnected, ideal subreactors is set up on the basis of the fermentor's configuration and flow pattern. The residence time distribution curve is used to test the validity of the model and the relative quantities of flow streams and regions in the model are determined. A least-square fitting procedure between measured and calculated distribution curves is used to identify the proper model. The application of this method to real cultivation conditions is also discussed. Using this approach, the multistage tower fermentor is shown to be equivalent to a cascade of four perfectly mixed tanks with a backtracking stream between stages. The extent of backflow under various conditions has also been determined.     

Selenium content of milk and milk products of Turkey. II

Selenium content of 1028 milk and milk products of Turkey are presented in this study. The selenium content of human milk (colostrum, transitional, and mature milk), various kinds of milk [cow, sheep, goat, buffalo, paper boxes (3%, 1.5%, 0.012% fat), bottled milk, condensed milk (10% fat), mineral added milk (1.6%), and banana, strawberry, and chocolate milk] and milk products (kefir, yogurt, Ayran, various cheese, coffee cream, ice cream, butter, margarine, milk powder, and fruit yogurt) in Turkey were determined by a spectrofluorometric method. The selenium levels of cow milks collected from 57 cities in Turkey were also determined. Selenium levels in cow milk varied with geographical location in Turkey and were found to be lowest for Van and highest for Aksaray. The results [milk (cow, sheep, goat, buffalo and human) and milks products] were compared with literature data from different countries.     

基因工程菌生产重组人组织因子的发酵研究

根据表达重组人组织因子基因工程菌的自身特点,在30L发酵罐上,通过控制发酵pH、葡萄糖浓度、诱导物磷酸浓度、搅拌速度及采用分批补料等方法,对重组人组织因子基因工程菌高密度发酵和高效表达的条件进行了研究。实验结果表明:诱导物终浓度低于0.1mmol/L,菌体密度OD60014,发酵周期10.5h,基因工程菌批发酵平均产量为37.41g/L,重组人组织因子表达量为6.56 mg/L。关键词:基因工程菌;高密度发酵;组织因子     

Optimum operating mode for a class of fermentation

This paper is concerned with optimization of the operating mode of a fermentor. Combining the various modes of operation—batch, semibatch, and continuous—the operating pattern which maximizes the desired metabolic product in a single fermentor is determined by using Kelley's transformation method with Pontryagin's maximum principle. Kelley's transformation method is a device which avoids the singular situation which occurs when the usual procedure of selecting the optimal control function by the maximum principle breaks down. This is the case in the problem considered in this paper. For lysine fermentation, the best operating mode depends on the fermentor capacity and operating time. The results of this study are summarized thus: (i) when the operating time is “long enough,” optimal conditions require that continuous operation follows either semibatch and/or batch operation, and (ii) when the fermentor capacity becomes “large enough,” semibatch operation becomes important.     

Influence of breed and husbandry on viscosity of Israeli goat milk yogurt

Goat’s milk yogurt prepared from milk of different goat breeds on different farm types resulted in different viscosity. Yogurt from milk of goat breeds browsing on pasture was richer in solids and resulted in significantly higher viscosity compared with that of cow milk yogurt and yogurt produced from milk of goats fed hay and concentrate indoors. Milk from goats browsing on pasture was diluted in total solids content close to that of milk from goats kept indoors and cows. Yogurt made from this diluted milk still resulted in higher viscosity. It appears that milk from goat breeds browsing on pastures contains certain factor(s) that causes an increase in the viscosity of yogurt produced from it.     

Utilization of Lactose, Glucose, and Galactose by a Mixed Culture of Streptococcus thermophilus and Lactobacillus bulgaricus in Milk Treated with Lactase Enzyme

The mechanism responsible for an increased rate of acid production when yogurt starter cultures are grown in milk treated with lactase enzyme was investigated by studying carbohydrate utilization and acid development by a pure culture of Streptococcus thermophilus and a mixed yogurt starter culture consisting of S. thermophilus and Lactobacillus bulgaricus. In milk containing glucose, galactose, and lactose, glucose and lactose (but not free galactose) were fermented. Fermentation of lactose in control milk was accompanied by the release of free galactose, with the result that carbohydrate utilization was less efficient than in treated milk. This phenomenon also occurred when lactose was fermented by S. thermophilus in broth culture. Carbohydrate utilization by the mixed yogurt culture was more rapid when the lactose in milk was partially prehydrolyzed. Our results suggest that the more rapid acid development that took place when a mixed yogurt starter culture was grown in milk containing prehydrolyzed lactose was the result of a more rapid and efficient utilization of carbohydrate by S. thermophilus when free glucose in addition to lactose was available for fermentation. The evidence presented also suggests that uptake and utilization of glucose and lactose by S. thermophilus are different in broth and milk cultures.     

The rotorfermentor. II. Application to ethanol fermentation.

The kinetics of microbial growth and product formation are described as applied to the high cell concentration scheme of the rotorfermentor. A bench scale pilot plant was designed and built in order to demonstrate the operational feasibility of the rotorfermentor. The fermentation of glucose to ethanol by Saccharomyces cerevisiae ATCC 4126 was used. When the rotorfermentor was used with a glucose feed concentration of 104 g/liter almost 100% glucose utilization was obtained and the ethanol productivity rate was 27.3 g ethanol/liter hr which was found to be about 10 times greater than the ethanol productivity obtained from an ordinary continuous stirred tank (CST) fermentor. The ethanol experimental results obtained from the rotorfermentor and an ordinary CST fermentor were used as a basis to assess the economic feasibility of the rotorfermentor. The economics of an industrial scale ordinary CST fermentor with and without cell recycle is compared with a rotorfermentor unit for the same ethanol production throughput. For the process conditions considered in this case, calculations showed that the rotorfermentor may replace both a CST fermentor and cell centrifuge resulting in lower capital equipment costs and lower power consumption requirements.     

Isolation and characterization of Lactobacillus delbrueckii ssp. bulgaricus and Streptococcus thermophilus from plants in Bulgaria

One of the traditional ways of preparation of yogurt starter in Bulgaria is placing a branch of a particular plant species into boiled sheep's milk maintained at about 45°C, which is further incubated until a dense coagulum is obtained. To investigate the possible origin of the yogurt starter bacteria, Lactobacillus delbrueckii ssp. bulgaricus ( L. bulgaricus ) and Streptococcus thermophilus ( S. thermophilus ), the traditional way of yogurt-starter preparation was followed. Hundreds of plant samples were collected from four regions in Bulgaria and incubated in sterile skim milk. The two target bacteria at low frequencies from the plant samples collected were successfully isolated. Phenotypic and genotypic characteristics of these bacterial isolates revealed that they were identified as L. bulgaricus and S. thermophilus . Twenty isolates of L. bulgaricus and S. thermophilus , respectively, were selected from the isolated strains and further characterized with regard to their performance in yogurt production. Organoleptic and physical properties of yogurt prepared using the isolated strains from plants were not significantly different from those prepared using commercial yogurt-starter strains.
It was therefore suggested that L. bulgaricus and S. thermophilus strains widely used for commercial yogurt production could have originated from plants in Bulgaria. To our knowledge, this is the first report on the isolation and characterization of L. bulgaricus and S. thermophilus strains from plants.     

Portal absorption of 15N and amino nitrogen in the growing pig after ingestion of labelled milk, yogurt or heat-treated yogurt.

The aim of this experiment was to study 15N and amino-nitrogen (AN) portal absorption in the growing pig after ingestion of uniformly (0.2509 APE) labelled 15N milk (M), yogurt ingested just after manufacturing (Y0), yogurt stored for 21 d at 4 degrees C (Y21) and heat-treated yogurt (HY). The highest porto-arterial differences (PAD) in 15N and AN were found in the period between 30 min and 90 min after ingestion. The absorption of nitrogen from M and HY mainly occurred during the 0-120 min time period (about 70% for M and 67% for HY). For Y0 and Y21, a larger displayed absorption period over the 0-240 min time period was observed. Y0 and Y21 presented a quite similar portal absorption profile. The 15N absorption rate was close to 80% for each studied milk product, suggesting that under our experimental conditions, dairy products (M, Y0, Y21 and HY) deliver nearly the same amounts of nitrogen to the organism. AN absorption rates were around 78% with a higher variability between the milk products. These results also indicate that most of the proteins were absorbed within the 240 min postprandial period.     

新型婴儿双歧杆菌马铃薯酸奶的工艺化初步研究

目的 以马铃薯和牛奶为主要原料,利用婴儿双歧杆菌进行发酵,制备一款新型酸奶。方法 采用婴儿双歧杆菌作为发酵菌株,发酵马铃薯牛奶。进行马铃薯酸奶原料的比例、酸奶发酵的温度和时间的探索。结果 当发酵温度42℃、发酵时间20 h、马铃薯汁10%时,酸奶的感官及口味较好。结论 利用10%马铃薯汁,牛奶等可初步制备出新型马铃薯汁酸奶。     

Growth of a Mixed Species Lactic Starter in a Continuous "pH-Stat" Fermentor

Optimum growth conditions for mixed species starter FDs 0172 in skim milk, whey medium, and tryptone medium at 25 C in a “pH-stat” continuous fermentor were investigated. Specific growth rate and productivity were found to be influenced both by the medium and the pH value. Highest productivity was found in skim milk medium between pH 5.5 and 5.9. Lactic acid-producing activity of harvested cells decreased by continuous cultivation at pH values lower than 5.9 to 6.1, especially in tryptone and whey medium. Bacterial balance, CO₂ production, and aroma formation were comparable to the control in skim milk and whey medium at any pH, but differed significantly for cells grown in tryptone medium.     

Enterotoxin production by Vibrio cholerae and Vibrio mimicus grown in continuous culture with microbial cell recycle.

We have examined the effect of complete cell recycle on the production of cholera toxin (CT) by Vibrio cholerae and CT-like toxin by Vibrio mimicus in continuous culture fermentations. Complete cell recycle was obtained by filtering culture fluids through Amicon hollow fibers with an exclusion limit of 100,000 daltons (H1P100-20) and returning the concentrated cell slurry to the fermentor. A single 1-liter laboratory fermentor system modified with this recycle loop was capable of producing over 20 liters of cell-free culture filtrate per day. Toxin production in this system was compared with yields obtained in traditional continuous cultures and in shake flask cultures. Yields of CT from V. cholerae 569B in the recycle fermentor were highest at the highest dilution rate employed (1.0 vol/vol per h). The use of complete cell recycle dramatically increased yields over those obtained in continuous culture and equaled those obtained in shake flasks. The concentration of CT in the filtrate was slightly less than half of that measured in culture fluids sampled at the same time. Similarly, V. mimicus 61892 grown in the presence of 50 micrograms of lincomycin per ml produced 280 ng of CT per ml in the recycle fermentor, compared with 210 ng/ml in shake flasks under optimal conditions. The sterile filtrate from this fermentation contained 110 ng/ml.     

A continuous,multistage tower fermentor. I. Design and performance tests

The design of a continuous column fermentor with a multiple staging effect is described. The column is divided into four compartments by horizontal perforated plates and is provided with a central agitator shaft driving an impeller in each compartment. A tube at the center of each plate forms a liquid seal around the shaft and also acts as a “downcomer.” The fermentor is normally operated with counter-current flow of gas and medium. Fresh medium is added to the top stage and product is withdrawn from the bottom. The effect of plate and agitator design on fermentor performance was studied in terms of factor such as oxygen transfer rate, gas holdup, and interstage mixing. By proper choice of the design parameters, the fermentor was made to approximate a perfect four-stage cascade in terms of reactor performance. Preliminary experiments were performed with air-water systems, but a more realistic picture of fermentor performance was obtained in experience involving propagation of Escherichia coli. Data for business and substrate concentrations in each stage confirmed the staging effect of the apparatus. The fermentor operated in a stable manner for periods of more than two weeks.     

Using Goat’s Milk,Barley Flour,Honey, and Probiotic to Manufacture of Functional Dairy Product

Stirred yogurt manufactured using probiotic culture which usually called Rayeb milk in the Middle East region is one of the most important functional fermented milk products. To increase the health and functionality properties to this product, some ingredients like fruits, cereal, and whey protein are used in production. This study was carried out to prepare functional Rayeb milk from goat’s milk, barley flour (15%) and honey (4%) mixtures using ABT culture. Also, vanilla and cocoa powder were used as flavorings. Adding barley flour and honey to goat’s milk increased curd tension and water-holding capacity and decreased coagulation time and susceptibility to syneresis. The values of carbohydrate, total solids, dietary fiber, ash, total protein, water soluble nitrogen, total volatile fatty acids, unsaturated fatty acids, oleic, linoleic, α-linolenic acids, and antioxidant activity were higher in Rayeb milk supplemented with barley flour and honey than control. The viabilities of Lactobacillus acidophilus and Bifidobacterium lactis Bb12 (Chr. Hansen’s Lab A/S) increased in fortified Rayeb milk. The recommended level of 10⁷ cfu g^?1 of bifidobacteria as a probiotic was exceeded for these samples. Addition of vanilla (0.1%) or cocoa powder (0.5%) improved the sensory properties of fortified Rayeb milk.     

Growth and activity of Bulgarian yogurt starter culture in iron-fortified milk

Bulgarian yogurts were manufactured and fortified with 8, 15 and 27 mg of iron kg(-1) of yogurt. The growth and acidifying activity of the starter culture bacteria Streptococcus thermophilus 13a and Lactobacillus delbrueckii subsp. bulgaricus 2-11 were monitored during milk fermentation and over 15 days of yogurt storage at 4 degrees C. Fortifying milk with iron did not affect significantly the growth of the starter culture during manufacture and storage of yogurt. Counts of yogurt bacteria at the end of fermentation of iron-fortified milks were between 2.1 x 10(10) and 4.6 x 10(10) CFU ml(-1), which were not significantly different from numbers in unfortified yogurts. In all batches of yogurt, the viable cell counts of S. thermophilus 13a were approximately three times higher than those of L. delbrueckii subsp. bulgaricus 2-11. Greater decrease in viable cell count over 15 days of storage was observed for S. thermophilus 13a compared to L. delbrueckii subsp. bulgaricus 2-11. Intensive accumulation of lactic acid was observed during incubation of milk and all batches reached pH 4.5 +/- 0.1 after 3.0 h. At the end of fermentation process, lactic acid concentrations in iron-fortified yogurts were between 6.9 +/- 0.4 and 7.3 +/- 0.5 g l(-1). The acidifying activity of starter culture bacteria in the control and iron-fortified milks was similar. There was no increase in oxidized, metallic and bitter off-flavors in iron-fortified yogurts compared to the control. Iron-fortified yogurts did not differ significantly in their sensorial, chemical and microbiological characteristics with unfortified yogurt, suggesting that yogurt is a suitable vehicle for iron fortification and that the ferrous lactate is an appropriate iron source for yogurt fortification.     

Performance of a two-stage fermentor with cell recycle for continuous production of lactic acid

The performasnce of a recycle two-stage fermentor with cell separators after each stage is analyzed numerically for continuous production of lactic acid. In this system, the bleed broth withdrawn from the first stage is provided to the second fermentor to reuse viable cells in the bleed. Biological rate expressions and parametric values are taken from the literature. The effects of operating parameters on the concentrations of total and viable cells, substrate and product in each stage, the lactic acid productivity and the substrate conversion are examined and discussed. With respect to overall productivity and conversion, it is found that the present fermentor system is more efficient than a conventional chemostat fermentor with cell recycle.