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.     

Effect of varying starch properties and mashing conditions on wort sugar profiles

The aim of this research was to investigate the relationship between starch composition in barley and its malted counterpart alongside malt enzyme activity and determine how these factors contribute to the fermentable sugar profile of wort. Two Australian malting barley varieties, Commander and Gairdner, were sourced from eight growing locations alongside a commercial sample of each. For barley and malt, total starch and gelatinisation temperature were taken, and for malt, α‐ and β‐amylase activities were measured. Samples were mashed using two mashing profiles (infusion and Congress) and the subsequent wort sugar composition and other quality measures (colour, original gravity, soluble nitrogen) were tested. Variety had no significant (<0.05) effect on any barley, malt, enzyme or wort characteristics. However, growing location impacted gelatinisation temperature, colour, malt protein content and original gravity. The gelatinisation temperature in malt samples was higher, by ~0.8°C, than in the equivalent barley sample. Several malt samples, even with protein contents <12.0%, had gelatinisation temperature >65°C. The fermentable sugars measured in the malt prior to mashing showed a higher proportion of maltose than glucose or maltotriose. In addition, there were significant differences in the amount of sugar produced by each mashing method with the high temperature infusion producing a higher amount of sugar and proportionally more maltose. There is scope for further research on the effect of genetics and growing environment on gelatinisation temperature, mash performance and fermentable sugar development. Routinely measuring gelatinisation temperature and providing this information on malt specification sheets could help brewers optimise performance. © 2019 The Institute of Brewing & Distilling     

Microbiota during fermentation of chum salmon (Oncorhynchus keta) sauce mash inoculated with halotolerant microbial starters: Analyses using the plate count method and PCR-denaturing gradient gel electrophoresis (DGGE)

Nine different combinations of mugi koji (barley steamed and molded with Aspergillus oryzae) and halotolerant microorganisms (HTMs), Zygosaccharomyces rouxii, Candida versatilis, and Tetragenococcus halophilus, were inoculated into chum salmon sauce mash under a non-aseptic condition used in industrial fish sauce production and fermented at 35 ± 2.5 °C for 84 days to elucidate the microbial dynamics (i.e., microbial count and microbiota) during fermentation. The viable count of halotolerant yeast (HTY) in fermented chum salmon sauce (FCSS) mash showed various time courses dependent on the combination of the starter microorganisms. Halotolerant lactic acid bacteria (HTL) were detected morphologically and physiologically only from FCSS mash inoculated with T. halophilus alone or with T. halophilus and C. versatilis during the first 28 days of fermentation. Only four fungal species, Z. rouxii, C. versatilis, Pichia guilliermondii, and A. oryzae, were detected throughout the fermentation by PCR-denaturing gradient gel electrophoresis (PCR-DGGE). In FCSS mash, dominant HTMs, especially eumycetes, were nonexistent. However, under the non-aseptic conditions, undesirable wild yeast such as P. guilliermondii grew fortuitously. Therefore, HTY inoculation into FCSS mash at the beginning of fermentation is effective in preventing the growth of wild yeast and the resultant unfavorable flavor.     

Production of an exopolysaccharide-containing whey protein concentrate by fermentation of whey

Using whey as a fermentation medium presents the opportunity to create value-added products. Conditions were developed to partially hydrolyze whey proteins and then ferment partially hydrolyzed whey with Lactobacillus delbrueckii ssp. bulgaricus RR (RR; an EPS-producing bacterium). In preliminary experiments, pasteurized Cheddar cheese whey was treated with Flavourzyme to partially hydrolyze the protein (2 to 13% hydrolyzed). Fermentation (2 L, 38 degrees C, pH 5.0) with RR resulted in EPS levels ranging from 95 to 110 mg of EPS per liter of hydrolyzed whey. There were no significant differences in the amount of EPS produced during fermentations of whey hydrolyzed to varying degrees. Since a high level of hydrolysis was not necessary for increased EPS production, a low level of hydrolysis (2 to 4%) was selected for future work. In scale up experiments, whey was separated and pasteurized, then treated with Flavourzyme to hydrolyze 2 to 4% of the protein. Following protease inactivation, 60 L of partially hydrolyzed whey was fermented at 38 degrees C and pH 5.0. After fermentation, the broth was pasteurized, and bacterial cells were removed using a Sharples continuous centrifuge. The whey was then ultrafiltered and diafiltered to remove lactose and salts, freeze-dried, and milled to a powder. Unfermented hydrolyzed and unhydrolyzed whey controls were processed in the same manner. The EPS-WPC ingredients contained approximately 72% protein and 6% EPS, but they exhibited low protein solubility (65%, pH 7.0; 58%, pH 3.0).     

Synthesis of galactooligosaccharides using sweet and acid whey as a substrate

Two commercial available lactases from Aspergillus oryzae and Kluyveromyces lactis were used to study the synthesis of galactooligosaccharides (GOS) in sweet and acid whey. At 38 g L⁻¹ initial lactose concentration, the A. oryzae enzyme gave a GOS yield of 10.91 ± 0.01% in lactose solution, 10.93 ± 0.18% in sweet whey and 11.32 ± 0.59% in acid whey. Thus, the components in whey did not influence the enzymes transgalactolytic activity. On the other hand, the K. lactis enzyme showed a strong dependence on whey type and whey concentration. At 38 g L⁻¹ initial lactose concentration, GOS yields were 10.93 ± 0.26% in lactose solution, 4.30 ± 0.17% in sweet whey and 10.56 ± 0.41% in acid whey. However, with increasing initial lactose concentration, the inhibitory effect of sweet whey was decreasing, which resulted in even higher yields than in lactose solution.     

Characterization of major volatile compounds in whey spirits produced by different distillation stages of fermented lactose-supplemented whey

This research aimed to advance the understanding of acceptable sensory qualities of potable whey-based spirit from nonsupplemented, mid-supplemented, and high-supplemented whey samples by analyzing major volatile compounds during different stages of distillation (head, heart, and tail). The results demonstrated that commercial Saccharomyces cerevisiae strain in lactase-hydrolyzed whey showed rapid and complete sugar hydrolysis and efficient ethanol production in 24, 30, and 36 h on average, producing up to 29.5, 42.1, and 56.4 g/L of ethanol, respectively. The variations in titratable acidity, specific gravity, pH value, residual protein, sugar content, and alcohol yield were investigated during the fermentation. The total amount of volatile compound concentrations significantly decreased from the head (2,087–2,549 mg/L) to the tail whey spirits (890–1,407 mg/L). In the whey spirit, 2-methyl-1-butanol, 3-methyl-1-butanol, 2-methyl-1-propanol, 1-propanol, acetaldehyde, and ethyl acetate were the most prevalent dominant compounds, accounting for the largest proportion of total volatile compounds. The volatile compounds detected were far below the acceptable legal limit. The results suggest that high sensory qualities of potable whey-based spirits can be produced by fermentation of lactose-supplemented whey with S. cerevisiae cells.     

Continuous Lactose Hydrolysis in Fixed-Bed and Expanded-Bed Reactors Containing Catalytic Resins

Kinetics studies of lactose hydrolysis in a continuous fixed-bed column reactor containing catalytic resins showed that the reaction was first order. The Q₁₀ value between 85 and 95°C was 4.08 and estimated activation energy was 36.9 Kcal/mole. Operational difficulties occurred when deproteinated whey was used as the substrate for the above process. Therefore, an expanded-bed process was developed to hydrolyze deproteinated whey. Acid was also added to the deproteinated whey to enhance the functionality of catalytic resin. The modified process improved reaction rate and facilitated process operation. At 95°C, 99% of the lactose in whey was hydrolyzed within 3 hr of residence time when the substrate was acidified with concentrated nitric acid to a final acid concentration of 0.6N.     

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。     

Effect of Cultural Conditions and Media on the Antimicrobial Activity of Streptococcus Thermophilus

When Streptococcus thermophilus was grown in basal medium with or without fermentable carbohydrate (lactose, glucose or sucrose at 2% level), antimicrobial activity was detected only in the cell-free medium and cells were free of any antimicrobial activity. Peanut milk, soymilk and basal medium with a fermentable sugar supported the production of antimicrobial compound(s) by S. thermophilus. Milk-based medium did not appear to be a necessity for such activity. When tested independently, the optimum temperature for maximum production of antimocrobial activity by S. thermophilus in whole milk was 50°C while the optimum time of incubation was 48 hr. An initial pH of 6.0 resulted in maximum antimicrobial activity in basal medium with 2% lactose.     

Ethanol from lactose in salted cheese whey by recombinant Saccharomyces cerevisiae

Seven yeast recombinants were selected from 57 fusant colonies on the basis of higher DNA content, nuclear diameter and ethanol yield compared to parental Saccharomyces cerevisiae ATCC 4126 and Kluyveromyces lactis CBS 683. Six recombinants out of the above colonies revealed growth on lactose and sucrose, indicating that they are S. cerevisiae with transformed β-galactosidase systems. The fusant colonies were investigated with respect to their capacity to convert lactose in salted cheese whey into ethanol. Among these recombinants that showed high tolerance towards sodium chloride and higher ethanol yield than lactose fermenting parental K. lactis CBS 683, SK-1 exhibited high tolerance up to 4 g dl^–1 sodium chlonride with an ethanol yield of 4.66 ml dl^–1 (v/v) , SK-23 tolerated 6 g dl^–1 sodium chloride with an ethanol yield of 4.14 ml dl^–1 (v/v) and SK-26 showed resistance towards 8 g dl^–1 sodium chloride and give an ethanol yield of 3 ml dl^–1 (v/v).     

Effect of germination and probiotic fermentation on nutrient composition of barley based food mixtures

Food mixtures formulated from non-germinated and germinated barley flour, whey powder and tomato pulp (2:1:1w/w) were autoclaved, cooled and fermented with 5% Lactobacillus acidophilus curd (10⁶ cells/ml) at 37 °C for 12 h. The cell count was found significantly higher (8.88 cfu/g) in the fermented food mixture formulated from germinated flour as compared to the non-germinated barley based food mixture. A significant drop in pH with corresponding increase in titratable acidity was found in the germinated barley flour based food mixture. Processing treatments like germination, autoclaving and probiotic fermentation did not bring about any significant change in ash and fat contents, but significant decrease was noticed in crude protein, crude fibre, starch, total and insoluble dietary fibre contents. The combined processing caused significant improvement in reducing sugar, thiamine, niacin, lysine and soluble dietary fibre contents of barley based food mixtures. In conclusion, a combination of germination and fermentation is a potential process for enhancing the nutritional quality of food mixtures based on coarse cereals.     

Proteolytic activity of Saccharomyces cerevisiae strains associated with Italian dry-fermented sausages in a model system

Strains of Saccharomyces cerevisiae isolated from Italian salami were screened for proteolytic activity in a model system containing sarcoplasmic (SMS) or myofibrillar (MMS) proteins, at 20 °C for 14 days, to evaluate the possible influence on the proteolysis of fermented sausages.SDS-PAGE revealed that 14 of the most osmotolerant strains were responsible for the extensive hydrolysis of the main myofibrillar proteins, while only one strain was able to hydrolyze sarcoplasmic proteins. Free amino acids (FAA) accumulated during proteolysis were strain-dependent with different patterns from sarcoplasmic or myofibrillar protein fraction. In general, proteolysis lead Cys, Glu, Lys and Val as the most abundant FAA in the inoculated MMS samples. Volatile compound analysis, determined by SPME-GC-MS, evidenced 3-methyl butanol in MMS, and 2-methyl propanol and 3-methyl-1-butanol in SMS as major compounds. Our findings highlight that S. cerevisiae could influence the composition in amino acids and volatile compounds in fermented sausages, with a strain-dependent activity.     

Monoterpene alcohols release and bioconversion by Saccharomyces species and hybrids

Terpene profile of Muscat wines fermented by Saccharomyces species and hybrid yeasts was investigated. The amount of geraniol decreased in most wines with respect to the initial must except for Saccharomyces bayanus wines. On the other hand, alpha-terpineol amount was higher in wines fermented by Saccharomyces cerevisiae and hybrid yeasts. The amount of linalool was similar in all wines and comparable to the amount in the initial must. Lower levels of beta-d-glucosidase activity were found in the hybrid yeasts with respect to S. cerevisiae. Moreover, no relationship between beta-d-glucosidase activity and terpenes profile in Muscat wines fermented with Saccharomyces species and hybrids was found. Growth of yeasts on minimum medium supplemented with geraniol showed bioconversion of geraniol into linalool and alpha-terpineol. Percentages of geraniol uptake and bioconversion were different between Saccharomyces species and hybrids. Strains within S. bayanus, Saccharomyces kudriavzevii and hybrids showed higher geraniol uptake than S. cerevisiae, whereas the percentage of produced linalool and alpha-terpineol was higher in S. cerevisiae and hybrid yeasts than in S. bayanus and S. kudriavzevii. The relationship between geraniol uptake and adaptation of Saccharomyces species to grow at low temperature is discussed.     

枸杞蜜酒的酿造工艺研究

以枸杞和蜂蜜为原料,用白砂糖调整发酵醪液的糖度,柠檬酸调整发酵醪液的酸度,筛选最适的酿酒活性干酵母,对枸杞蜜酒的酿造工艺进行研究。通过正交试验确定发酵的最佳工艺条件为枸杞浸提汁与蜂蜜配比为19∶1,发酵醪液的起始糖度240 g/L,柠檬酸调pH值至3.8,在温度为24℃±2℃条件下进行发酵,发酵至第4天时补加白砂糖80 g/L,用0.15 g/L的明胶和2.0 g/L的膨润土配合使用对枸杞蜜酒进行澄清稳定化处理,得到的枸杞蜜酒呈金黄色,具有枸杞和蜂蜜特殊的香气且酒体醇厚、酸甜协调。     

Influence of malted barley and exogenous enzymes on the glucose/maltose balance of worts with sorghum or barley as an adjunct

The sugar profile of wort from laboratory malted barley, malted sorghum, unmalted barley and unmalted sorghum grains mashed with commercial enzyme preparations were studied. Similar levels of glucose to maltose (1:7) were observed in wort of malted barley and malted sorghum. Mashing barley or sorghum grains with commercial enzymes changed the glucose to maltose ratio in both worts, with a greater change in wort from sorghum grains. Although hydrolysis with commercial enzymes released more glucose from maltose in sorghum wort, the same treatment retained more maltose in barley wort. Adding malted barley to sorghum grains mashed with commercial enzymes restored the glucose to maltose ratio in sorghum mash. Fermentation of wort produced from all barley malt (ABM) mash and commercial enzyme/barley malt/sorghum adjunct (CEBMSA) mash of similar wort gravity was also studied. ABM and CEBMSA worts exhibited similar glucose to maltose ratios and similar amino acid spectra. However, ABM released more individual amino acids and five times more proline than wort from commercial enzyme/barley malt/sorghum adjunct. ABM produced 27% more glucose and 7% more maltose than CEBMSA. After fermentation, ABM mash produced 9.45% ABV whilst commercial enzyme/barley malt/sorghum adjunct mash produced 9.06% ABV. Restoration of the glucose/maltose ratio in the CEBMSA mash produced wort with a sugar balance required for high gravity brewing. © 2020 The Institute of Brewing & Distilling     

Prebiotic effects of fermentable carbohydrate polymers may be modulated by faecal bulking of non‐fermentable polysaccharides in the large bowel of rats

This study investigated the impact of supplementary fermentable polysaccharides inulin (a prebiotic) and barley β‐glucan on the large bowel, when consumed against a background high or low intake of mixed non‐starch polysaccharide. Rats were fed for 28 days with diets containing high (15%) and low (5%) background dietary fibre (BDF; cellulose plus sugar beet fibre), with or without 5% supplementary fermentable fibre (SFF; inulin or barley β‐glucan). High‐BDF diets unsupplemented with inulin or barley β‐glucan caused greater improvement than supplemented low‐BDF diets in several parameters linked to gut health: they increased colonic Bifidobacterium spp., butyric acid concentration in the caecum, and colonic crypt depth more than inulin and barley β‐glucan. For these parameters, inulin and barley β‐glucan added little to the effects of the high‐BDF diet alone. Furthermore, by allowing for dilution due to the greater faecal bulk induced by the mixed fibre compared with inulin or barley β‐glucan, the total quantity of butyric acid in the colon of BDF‐fed rats was greatly amplified. Interpretation of the effects of prebiotics in diets containing dietary fibre requires concurrent analysis of the multiple effects of non‐fermentable bulk.     

Validation and application of osmolyte concentration as an indicator to evaluate fermentability of wort and malt

The objective was to develop a new simple and quick approach to predict fermentability, based on osmolyte concentration (OC). Eight malts were assayed for diastatic power, starch‐degrading enzymes [α ‐amylase, β ‐amylase and limit dextrinase (LD)] and malt OC (MOC). All malts were mashed to determine wort OC (WOC), real degree of fermentation (RDF) and sugar contents in a small‐scale mashing protocol. The results showed that MOC was correlated with malt α ‐amylase, LD, the resultant WOC, RDF and fermentable sugar (r  = 0.813, 0.762, 0.795, 0.867, 0.744, respectively), suggesting that MOC was discriminating in predicting levels of malt amylolytic enzymes, wort sugar and RDF without the mashing and fermentation process. Moreover, WOC showed stronger correlations with malt α ‐amylase, LD, RDF and fermentable sugars (r  = 0.796, 0.841, 0.884, 0.982, respectively), suggesting that WOC can be used to quickly predict wort sugar contents and RDF without a fermentation step. Furthermore, the effects of mashing temperature and duration on WOC, RDF and sugar contents are discussed. Adjusting mash temperature to 65°C or extending the mash duration dramatically increased RDF and WOC, whereas malt extract was relatively stable. Similarly, WOC showed significant correlations with RDF and fermentable sugars (r  = 0.912 and 0.942, respectively), suggesting that WOC provides a simple and reliable tool to assist brewers to optimize mash parameters towards the production of ideal wort fermentability. In conclusion, the ability of OC to predict malt fermentability and sugar content allows brewers to keep better control of fermentability in the face of variation of malt quality, and to quickly adjust mashing conditions for the consistency of wort fermentability. Copyright © 2017 The Institute of Brewing & Distilling     

Removing gluconic acid by using different treatments with a Schizosaccharomyces pombe mutant: Effect on fermentation byproducts

Three different treatments involving inoculation with Schizosaccharomyces pombe YGS-5 and Saccharomyces cerevisiae G1 strains were tested with a view to reducing the amount of gluconic acid in synthetic medium. The treatments involved (a) simultaneous inoculation with S. cerevisiae and S. pombe (SpSc); (b) depletion of gluconic acid with S. pombe and subsequent inoculation with S. cerevisiae following removal of S. pombe from the medium (Sp − Sp + Sc); and (c) as (b) but without removing S. pombe from the medium (Sp + Sc). The results thus obtained were compared with those for a control treatment involving fermentation with S. cerevisiae alone (Sc). The amounts of volatile compounds quantified in the fermented media were similar with the treatments where gluconic acid was previously depleted (viz.Sp − Sp + Sc and Sp + Sc). Amino acids were used in large amounts by S. pombe during removal of gluconic acid; this affected subsequent fermentation by S. cerevisiae and the formation of byproducts. Based on the gluconic acid uptake, fermentation kinetics, volatile composition and absence of off-flavours in the fermented media, both treatments (Sp − Sp + Sc and Sp + Sc) can be effectively used in winemaking processes to remove gluconic acid from must prior to fermentation.     

Saccharification and Fermentation of Whole Barley Ground in the Szego Mill

Barley, after steeping in water, was ground with ease and efficiency in the Szego mill, and its starch was liquefied, saccharified and fermented to very high yields of ethanol. The Szego mill consists of vertical rollers with helical grooves which rotate within a fixed cylinder, resulting in very fine grinding and a somewhat flaky product. The steeped barley was ground to a fine paste. This was readily liquefied and saccharified by amylolytic enzymes (dual enzyme process), and the resulting sugars were fermented in 24 h by ordinary bakers' yeast Saccharomyces cerevisiae, resulting in over 450 1 ethanol/t of barley. Still shorter time, 12 h, and the same high yield were achieved when liquefied barley starch was simultaneously saccharified by glucoamylase and fermented. Fermentation to ethanol by a glucoamylase-producing yeast S. diastaticus strain 164 A (from Labatt Brewing Company) enabled the amount of this enzyme required for saccharification to be reduced to about one-half the normal quantity, but at some cost in slower fermentation and slightly lower ethanol yield.