A recombinant Saccharomyces cerevisiae strain for efficient conversion of lactose in salted and unsalted cheese whey into ethanol

For utilization of lactose in salted and unsalted cheese whey, intergeneric protoplast fusion between lactose nonfermenting, salt-tolerant Saccharomyces cerevisiae ATCC4126 and lactose fermenting Kluyveromyces lactis CBS683 was carried out. The fusion process gave rise to new hybrid yeast strains that revealed higher significant DNA contents than parental strains. The recombinants showed growth on either lactose or sucrose. The ethanol yields by some recombinants were 5.55% from sweet whey and 4.66% from salted whey containing up to 6% sodium chloride compared to 4.15 and 2.86% for parental K. lactis CBS683, respectively.     

Inhibition of toxicogenic Bacillus licheniformis 553/1 in Nigerian Wara soft cheese by nisin-producing Lactococcus lactis LAC309

Wara soft cheese is a traditionally produced cheese in Nigeria. The production of this cheese includes a heating step for killing vegetative cells. Therefore, mainly spore-forming bacteria surviving the process may function as the first spoilers resulting in lowered shelf-life and safety of the product. In this study, we investigated if the addition of a nisin-producing Lactococcus lactis LAC309 starter after the high temperature treatment could result in nisin production and inhibition of the toxicogenic Bacillus licheniformis 553/1 strain spiked into the cheese. The results showed that L. lactis LAC309 could produce nisin in Wara cheese and that the strain inhibited (3 log reduction) B. licheniformis 553/1 in Wara cheese. Food technologists developing industrialised Wara cheese should therefore consider including a nisin-producing starter strain in the cheese-making process.     

STABILIZATION OF THE ACTIVITY OF β-GALACTOSIDASE IN PERMEABILIZED IMMOBILIZED CELLS FOR HYDROLYSIS OF LACTOSE IN MILK

A potentially low cost β‐galactosidase was prepared as a crude permeabil‐ized cell mass of the yeast Kluyveromyces lactis that had been grown in ultrafiltered cheese whey. The enzyme preparation was immobilized in alginate beads. Milk lactose hydrolysis rates were 25% higher in manganese alginate beads than in calcium alginate beads. The immobilized biocatalyst lost activity when stored in calcium chloride solution, however, storage in 5 mM DTT or 50% glycerol allowed the biocatalyst to be recycled at least 5 times without any loss of activity.     

Effect of carbon dioxide under high pressure on the survival of cheese starter cultures

A new processing method that rapidly forms curds and whey from milk has the potential to improve cheesemaking procedures if cheese starter cultures can tolerate the processing conditions. The survival of Lactobacillus delbrueckii ssp. bulgaricus, Lactococcus lactis ssp. lactis, or Streptococcus thermophilus through this new process was evaluated. Inoculated milk containing 0, 1, or 3.25% fat or Lactobacillus MRS broth or tryptone yeast lactose broth (depending on microorganism used) was sparged with CO2 to a pressure of 5.52 MPa and held for 5 min at 38 degrees C. Broth contained 7.93 to 8.78 log CFU/ ml before processing and 7.84 to 8.66 log CFU/ml afterward. Before processing, milk inoculated with L bulgaricus, L. lactis, or S. thermophilus contained 6.81, 7.35, or 6.75 log CFU/ml, respectively. After processing, the curds contained 5.68, 7.32, or 6.50 log CFU/g, and the whey had 5.05, 6.43, or 6.14 log CFU/ml, respectively. After processing, the pHs of control samples were lower by 0.41 units in broth, 0.53 units in whey, and 0.89 units in curd. The pH of the processed inoculated samples decreased by 0.3 to 0.53 units in broth, 0.32 to 0.37 units in whey, and 0.93 to 0.98 units in the curd. Storing curds containing L. lactis at 30 degrees C or control curds and curds with L. bulgaricus or S. thermophilus at 37 degrees C for an additional 48 h resulted in pHs of 5.22, 5.41, 4.53, or 4.99, respectively. This study showed that milk inoculated with cheese starter cultures and treated with CO2 under high pressure to precipitate casein-produced curds that contained sufficient numbers of viable starter culture to produce lactic acid, thereby decreasing the pH.     

发酵型牦牛乳清酒的工艺优化

为实现奶酪生产副产物乳清的资源化利用,采用两段式发酵工艺及响应面优化确定了一种发酵型牦牛乳清酒制备工艺。以醪液酒精度为响应值,发酵温度、总接种量、发酵时间及初始pH为因素采用Box-Behnken设计建立数学模型;检测成品酒中各理化指标及氨基酸、有机酸含量并进行品评分析。结果表明:采用先接种乳酸克鲁维酵母后接种酿酒酵母工艺,乳酸克鲁维酵母发酵54 h后,醪液中乳糖含量为0.8%,利用率达到92.7%;酿酒酵母在发酵温度30 ℃、总接种量8%（乳酸克鲁维酵母4%）、发酵时间70 h、初始pH5.5条件下,可获得酒精度为14.1%Vol的牦牛乳清酒;酒液各项理化指标符合国家标准,必需氨基酸、总氨基酸及有机酸含量分别提高了0.7、1.1、0.8倍,是一种营养丰富的奶酒。     

Production of Beta-D-Galactosidase from Kluyveromyces fragilis Grown in Cheese Whey

Two strains of Kluyveromyces fragilis (145 and 276) and one of Kluyveromyces lactis were tested for their abilities to produce beta-D-galactosidase in cheese whey. Kluyveromyces fragilis 145 was selected for its higher beta-D-galactosidase activity per cell at the end of the exponential growth phase.Addition of ammonium sulfate (.3%) and yeast extract (.1%) to the deproteinized cheese whey increased cell mass and enzyme yield.Addition of 3% lactose did not affect beta-D-galactosidase activity per cell, which responded positively to a reduction in aeration from 1 to .25 air volume/medium volume/min.The harvested yeast cells were ethanol- and acetone-permeabilized to enhance lactose hydrolysis by beta-D-galactosidase.     

Development and Evaluation of Whole-Cell Yeast Lactase for Use in Dairy Processing

A low-cost industrial grade lactase was developed for one-time use in dairy products. The preparation contained the entire biomass of a selected hyperproducing strain of the yeast Kluyveromyces fragilis. Intracellular lactase was made freely accessible to its substrate by permeabilization of the cell membrane with food compatible reagents. In 0.1M phosphate plus 0.4% methyl paraben, permeabilization was complete in 0.5–1 hr at 50°C, with 90% activity recovery from 180 g/L of cells. Whole-cell lactase contained no viable cells and was free of proteolytic activity. Its pH optimum of 5.6–6.0 proved suitable for lactose hydrolysis concurrent with cottage cheese fermentation. Hydrolyzed whey was used in ice cream and bakers' yeast production.     

Fermentative Activity of Baker's Yeast Cultivated on Cheese Whey Permeate

Fermentative characteristics of Baker's yeast (Saccharomyces cerevisiae) produced on two cheese whey ultrafiltrate media (one in which lactose was hydrolyzed and one pre-fermented by Streptococcus thermophilus) were compared to those of yeast produced on molasses. Fermentative activity of yeast grown on molasses was superior to that of cells produced in whey. Addition of sucrose to pre-fermented whey ultrafiltrate (FWU) in the final stage of the fermentation increased fermentative activity. Fermentative activity of three strains grown on FWU varied considerably, certain strains being better adapted to the whey media. Breads produced with yeast grown on FWU were identical to those obtained with cells grown on molasses.     

Short communication: Conversion of lactose and whey into lactic acid by engineered yeast

Lactose is often considered an unwanted and wasted byproduct, particularly lactose trapped in acid whey from yogurt production. But using specialized microbial fermentation, the surplus wasted acid whey could be converted into value-added chemicals. The baker’s yeast Saccharomyces cerevisiae, which is commonly used for industrial fermentation, cannot natively ferment lactose. The present study describes how an engineered S. cerevisiae yeast was constructed to produce lactic acid from purified lactose, whey, or dairy milk. Lactic acid is an excellent proof-of-concept chemical to produce from lactose, because lactic acid has many food, pharmaceutical, and industrial uses, and over 250,000 t are produced for industrial use annually. To ferment the milk sugar lactose, a cellodextrin transporter (CDT-1, which also transports lactose) and a β-glucosidase (GH1-1, which also acts as a β-galactosidase) from Neurospora crassa were expressed in a S. cerevisiae strain. A heterologous lactate dehydrogenase (encoded by ldhA) from the fungus Rhizopus oryzae was integrated into the CDT-1/GH1-1–expressing strain of S. cerevisiae. As a result, the engineered strain was able to produce lactic acid from purified lactose, whey, and store-bought milk. A lactic acid yield of 0.358 g/g of lactose was achieved from whey fermentation, providing an initial proof of concept for the production of value-added chemicals from excess industrial whey using engineered yeast.     

Sugar–cellulose composites. VI. Economic evaluation of lactose production from cheese whey for use in paper

Previous papers in this series have shown that a portion of the cellulose fibres in paper can be replaced by lactose without significant changes in the physical properties of the paper. Lactose will only be used to substitute up to 10% of cellulose in paper‐making if it is available in quantity at a price less than that of the fibres replaced. Cheese is produced worldwide in large quantities, and its by‐product, whey, is mainly dried or, in some cases, fractionated to produce lactose and whey protein concentrate (WPC). Lactose obtained from cheese whey for this application does not need to fulfil FDA or EU requirements. Hence its manufacture is cheaper than for edible grade lactose. Furthermore, it is not necessary to dry the cellulose substitute to minimise the transportation cost if the lactose fractionation plant is located close to the paper‐mill. An analytical case study has been developed for the industrial environment existing in Asturias (Spain), where seven plants producing more than 400 000 t year^?1 of cheese whey, with an annual growth of 3%, and a 150 000 t year^?1 paper‐mill are located within a circular area of 80 km radius. This analysis has been extended to different plant capacities to allow its application to other locations with similar characteristics. © 2002 Society of Chemical Industry     

Whey as a source of peptides with remarkable biological activities

The dairy industry generates increased amounts of whey from both cheese and casein production facilities. Whey presents an elevated content of lactose and proteins, which are associated with its high biological oxygen demand and decomposing potential. Despite its potential as pollutant, whey has been considered as a dairy by-product due to its nutritional, functional and bioactive properties. The use of enzyme technology may be an interesting strategy to convert whey into added-value products. The hydrolysis of whey proteins can generate bioactive peptides, which are described to perform physiological effects in vivo, such as antioxidant, antimicrobial, antihypertensive and antidiabetic activities. Bioactive peptides derived from whey proteins have been also associated with immunomodulatory, anticancer, opioid and hypocholesterolemic activities. This review presents a discussion on the main biological activities of peptides derived from whey proteins.     

Detection and identification of Lactobacillus delbrueckii subsp. lactis bacteriophages by PCR

A sensitive PCR method amplifying an internal fragment of the major tail protein gene was developed to detect Lactobacillus delbrueckii subsp. lactis lytic bacteriophages in undefined, thermophilic whey starters used in Italy for production of Grana and Provolone cheeses. PCR was applied to several lytic Lb. delbrueckii subsp. lactis bacteriophages, which were highly diverse according to restriction analysis and phage host range. PCR detected the presence of phages in two out of 11 cultures, when applied to whey starters for Grana Padano cheese sampled from different cheese plants. The presence of actively growing phages in infected cultures was confirmed by traditional test. The PCR method proved to be useful to screen for the presence of Lb. delbrueckii subsp. lactis phages in thermophilic whey starters.     

Growth stimulation of a proteinase positive Lactococcus lactis strain by a proteinase negative Lactococcus lactis strain

Lactococcus lactis AMP15/pAMP31(D471R) is a proteinase negative, lactose negative strain with a modified oligopeptide transport system, and potential as a debittering agent due to its efficient utilization of hydrophobic peptides. Five wild L. lactis strains of dairy origin, which produced cheeses of high flavour quality, were cocultured with L. lactis AMP15/pAMP31(D471R) in an attempt to select adequate combinations of strains for use as defined cheese starters with potential debittering ability. Four of these strains, L. lactis B6, K16, M21 and P21, inhibited growth of L. lactis AMP15/pAMP31(D471R) at a level of 10(6) to 10(7) cfu mL(-1) after 24 h of incubation, even though production of bacteriocin-like compounds could only be proven for L. lactis M21. When L. lactis AMP15/pAMP31(D471R) was cocultured with the fifth strain, L. lactis N22, its growth was significantly (P<0.001) inhibited whereas growth of L. lactis N22 was significantly stimulated. The nature of the interaction was studied and it was established that L. lactis N22 is auxotrophic for folate, a compound produced and excreted by L. lactis AMP15/pAMP31(D471R).     

Construction of an enzymatic route using a food-grade recombinant Bacillus subtilis for the production and purification of epilactose from lactose

Lactose is a main by-product in the cheese industry. Many attempts have been made to convert the lactose to high value-added products, including epilactose. Epilactose is a valuable prebiotic and can be epimerized from lactose with cellobiose 2-epimerase (CEase). The objective of the present work was to construct a food-grade recombinant Bacillus subtilis that produces CEase from Thermoanaerobacterium saccharolyticum. The CEase was expressed in B. subtilis without antibiotic resistance genes. After fermentation, the maximum volumetric activity of the fermented broth was more than 7 U/mL. The activity of the recombinant B. subtilis was increased by up to 3.7 fold after ethanol permeabilization. Then, 66.9 ± 0.7 g/L of epilactose was produced from 300 g/L of whey powder solution in 1 h with 13.3 U/mL of permeabilized biocatalyst. In addition, an enzymatic route including degradation of the lactose, yeast fermentation, and cation exchange chromatography was described to further purify the produced epilactose from lactose. Finally, epilactose with a purity >98% was produced from 300 g/L of lactose with a yield of 24.0%. In conclusion, neither antibiotics nor pathogenic bacteria were used throughout the epilactose production and purification procedure.     

新疆塔城地区哈萨克族传统奶酪中酵母菌的分离鉴定

为保护新疆哈萨克族传统奶酪中的优良酵母菌株,从新疆塔城牧区不同牧场采集的10份哈萨克族传统奶酪样品中,分离得到44株酵母菌。采用形态学、生理生化特性鉴定、5.8S rDNA序列同源性分析相结合的方法,对分离菌株进行鉴定。共鉴定出5个种,其中34株库德毕赤酵母（Pichia kudriavzevii）,为优势菌株,6株戴尔有孢圆酵母（Torulaspora delbrueckii）,2株乳酸克鲁维酵母（Kluyve- romyces lactis ）,1株马克思克鲁维酵母（Kluyveromyces marxianus ）,1株发酵毕赤酵母（Pichia fermentans ）。结果表明,哈萨克族传统奶酪制品中所含酵母菌与其他地区的存在差异性,有其独特的酵母菌资源。     

Highly efficient assimilation of lactose by a metabolically engineered strain of Saccharomyces cerevisiae

A diploid strain of Saccharomyces cerevisiae able to metabolize lactose with high efficiency has been obtained. Haploid strains of Saccharomyces able to grow on lactose were constructed by cotransformation with two genes of Kluyveromyces lactis required for the utilization of the sugar, LAC4 and LAC12, encoding β-galactosidase and lactose permease respectively. Both genes were placed under the control of a galactose-inducible promoter and targeted to the rDNA encoding region (RDN1 locus) of the Saccharomyces genome. Lac⁺ transformants were selected on medium with lactose as the only carbon source. These transformants were mitotically stable, they maintained the Lac⁺ phenotype after growing in non-selective medium for more than 60 generations, but their growth was slow. We found that this lack of vigour was caused by their genetic background and not by a deficient expression of the heterologous genes. Therefore, their performance could be improved by crossing with a wild-type strain. Among the offspring of the crosses, two strains of opposite mating type were selected and mated to obtain a fast-growing Lac⁺ diploid. This diploid strain showed the typical fermentative behaviour of S. cerevisiae when it was grown in aerated liquid medium with glucose. In lactose medium, it exhibited a respiro-fermentative metabolism similar to that of K. lactis, with low ethanol production and high biomass yield. © 1998 John Wiley & Sons, Ltd.     

Selection and use of autochthonous multiple strain cultures for the manufacture of high-moisture traditional Mozzarella cheese

Lactobacillus plantarum 18A, Lactobacillus helveticus 2B, Lactobacillus delbrueckii subsp. lactis 20F, Streptococcus thermophilus 22C, Enterococcus faecalis 32C and Enterococcus durans 16E were the most acidifying strains within 146 isolates for natural whey starters. The effect of media and temperature on 2 autochthonous multiple strain cultures (AMSI: 18A, 2B, 20F and 22C, 32C and 16E and AMSII: 18A, 2B, 20F and 22C) was studied. Genomic analysis showed a constant cell numbers for AMSII during 16 days of propagation in whey milk. Mozzarella cheese was made by using AMSII, commercial starter (CS) or citric acid (DA). Compared to other cheeses, the DA had a lower level of protein, ash, Ca, free amino acids and a higher level of moisture. Based on confocal laser scanning microscopy analysis, AMSII cheese showed the lowest microstructural variations during the period of storage compared to other cheeses. All the sensory attributes were scored highest for AMSII cheese. ASMII extend the shelf-life to ca. 12-15 days instead of the 5-7 days of traditional high-moisture Mozzarella cheese.     

Characterization of yeasts involved in the ripening of Pecorino Crotonese cheese

The aims of this work were to identify and characterize for some important technological properties the yeast species present throughout the ripening process of Pecorino Crotonese, a traditional cheese produced in a well defined area of Southern Italy. In particular, the strain technological properties considered include fermentation/assimilation of galactose and lactose, assimilation of lactate and citrate in the presence of different NaCl concentrations, hydrolysis of butter fat, skim milk, gelatine and casein, production of brown pigments in cheese agar and ability to produce biogenic amines. High yeast levels were recorded in cheese samples already after 5 h of brining (about 5 log cfu/g) and these concentration remained constant during ripening. The yeast isolates belonged to restrict number of yeast species. While Kluyveromyces lactis and Saccharomyces cerevisiae were isolated prevalently in the first stages of Pecorino Crotonese production, Yarrowia lipolytica and Debaryomyces hansenii dominated during the later stages of maturation. Otherwise, the latter two were very NaCl resistant species. In fact, D. hansenii strains conserved the ability to assimilate lactose and galactose in the presence of 10% NaCl, while almost all the strains of Y. lipolytica isolated assimilated citrate and lactate up to 7.5% NaCl. Y. lipolytica isolates evidenced also the highest proteolytic and lipolytic activities and the capability to catabolize tyrosine producing brown pigment. In addition they resulted in the highest aminobiogenic potential decarboxylating ornithine, phenylalanine, tyrosine and lysine. However, they were not able to produce histamine, biogenic amine produced by three strains of D. hansenii.     

Milk sugars and minerals as ingredients

Lactose is the natural carbohydrate source and prebiotic compound found in the milk of mammals, but large variations in lactase activity in the small intestines of adult populations can cause problems with its use. The value of lactose can be increased by hydrolysis, but even more valuable products can be made by changing the structure of lactose and preventing its absorption in the gut. Some of these nonabsorbable lactose derivatives are already used in medical and functional food applications.
Calcium phosphate precipitation to the heat-transfer surfaces is one of the oldest problems of the dairy industry, but if precipitation is carried out in controlled conditions, the precipitate can be further processed to form milk calcium powder. Milk calcium can be used as a natural source of calcium in calcium-fortified dairy products.
The mineral and salty taste of whey has reduced its use as a food ingredient. The use of modern membrane technology offers a means of producing whey salt as a by-product of whey demineralization. These otherwise wasted minerals can then be used as a natural mineral salt. Especially interesting is the possibility of recycling the whey salt into cheese, improving its nutritional status.     

Whey Wine from Concentrates of Reconstituted Acid Whey Powder

A process for making table whey wine is based on fermenting with lactose-fermenting yeast, high-lactose (18 to 25%) whey concentrates, or permeates from reconstituted cottage cheese acid-whey powder. Deproteinization of the whey concentrates which gave a clear fermentation substrate that was rich in lactose was attained through ultrafiltration at about 40 C. Salts were reduced by electrodialysis to levels permitting optimum fermentation of sugar to alcohol. Active fermentation occurred at 25 to 30 C during 5 to 7 days.The table whey wine had 10% (vol/ vol), or more, alcohol that was derived wholly from lactose. After the finishing step, the fresh wine was clear, pale yellow, and had a pleasing tart taste and bouquet free from whey flavor. Its body was full.