Reduction of Lactococcus lactis phage contamination in whey by means of membrane filtration: Impact of phage morphology and of bacterial host cells functioning as “phage fishing tool”

For dairy bacteriophages, the impact of phage morphology and whey components on phage retention efficiencies in filtration processes is not known. Therefore, the influence of (i) five differently shaped Lactococcus lactis phages as well as (ii) differently treated bacterial host cells added to native whey were analysed via cross-flow membrane filtration experiments with polyethersulfone membranes. Filtration efficiency with respect to phage retention and whey protein permeation was investigated. Compared with untreated native whey, phage retention was significantly higher (up to 4.8 log units) in the experiments using whey that was spiked with mechanically disrupted lactococcal host cells. However, the retention of phages commonly found in the dairy environment (i.e., members of the 936, c2, and P335 phage groups) did not correlate with the phage morphology. Results of this model study clearly demonstrate that phage titres can be reduced in whey (containing a sufficient protein concentration) by flat sheet membrane filtration processes.     

Production of phage free cheese whey: Design of a tubular laboratory membrane filtration system and assessment of a feasibility study

An interposed “phage filtration” process step is proposed prior to subsequent processing of cheese whey to whey products to reduce fermentation risks due to phage contamination. At laboratory scale, the characteristics of three single tube ceramic membranes on the filtration performance in terms of phage retention, protein transmission and filtrate flux were analysed. From these tested membranes, a 0.1 μm microfiltration membrane was found to be suitable for significant phage reduction (3.4 log units) in cheese whey, simultaneously allowing total protein transmission of 56.2%. The experiments within the designed feasibility study conducted with a pilot plant microfiltration system provided a higher phage retention (4.1 log units) and a significantly increased transmission of major whey proteins (up to 84%) in comparison with the laboratory plant. The results shown in this study can easily be adapted by the dairy industry for better phage control and enhanced process safety.     

Influence of the suspension media on the thermal treatment of mesophilic lactococcal bacteriophages

The thermal resistances of the Lactococcus lactis phages P008 (a heat-sensitive wide-spread phage) and P680 (a heat-resistant phage) suspended in milk and in solutions supplemented with milk components were studied to elucidate the protective role of milk on phage inactivation. For both phages a decrease of inactivation was observed in casein solution. Furthermore, the inactivation kinetics of the phages in whey, in whey cream (3.5%, 20%, 30% fat) and in whey protein concentrate (0.7%, 5%, 10% protein) were tested. The inactivation experiments in whey cream and in whey protein concentrate having different concentrations of fat and protein revealed that fat had no influence on the inactivation, while, in contrast, the presence of protein had a protective effect.     

The distribution of lactococcal bacteriophage in the environment of a cheese manufacturing plant

Survival of starter bacteria and their bacteriophages following spray irrigation of whey in the vicinity of a cheese plant was investigated. Phage in environmental samples that attacked starter strains used in the plant were detected infrequently in water, but persisted for up to six weeks in the soil. Enrichment of cow dung, grass and trough water samples was required to detect phage, indicating their presence at low levels. No starter bacteria could be re-isolated from grass samples from whey irrigated pasture. Inside the plant, whey contained the largest numbers of phage and when released into the environment (via irrigation practices) contributed to the recycling of phages through the plant. It did not, however, appear to be the initial source for starter infection by ‘new’ phage. The detection of phages in the bulk raw milk that were not active on starter strains that were being used in the plant, reemphasized raw milk as a potential source of new phage.     

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.     

Whey filtration: a review of products,application, and pretreatment with transglutaminase enzyme

In the cheese industry, whey, which is rich in lactose and proteins, is underutilized, causing adverse environmental impacts. The fractionation of its components, typically carried out through filtration membranes, faces operational challenges such as membrane fouling, significant protein loss during the process, and extended operating times. These challenges require attention and specific methods for optimization and to increase efficiency. A promising strategy to enhance industry efficiency and sustainability is the use of enzymatic pre-treatment with the enzyme transglutaminase (TGase). This enzyme plays a crucial role in protein modification, catalyzing covalent cross-links between lysine and glutamine residues, increasing the molecular weight of proteins, facilitating their retention on membranes, and contributing to the improvement of the quality of the final products. The aim of this study is to review the application of the enzyme TGase as a pretreatment in whey protein filtration. The scope involves assessing the enzyme's impact on whey protein properties and its relationship with process performance. It also aims to identify both the optimization of operational parameters and the enhancement of product characteristics. This study demonstrates that the application of TGase leads to improved performance in protein concentration, lactose permeation, and permeate flux rate during the filtration process. It also has the capacity to enhance protein solubility, viscosity, thermal stability, and protein gelation in whey. In this context, it is relevant for enhancing the characteristics of whey, thereby contributing to the production of higher quality final products in the food industry. © 2023 Society of Chemical Industry.     

Characterization of lactococcal bacteriophages isolated from Slovenian dairies

Summary This is the first study on lactic acid bacteria bacteriophages isolated from dairy plants in Slovenia. Over a period of 2 years, thirteen lactococcal phages were isolated from the whey samples taken in three dairy plants. Phages were characterized by morphological properties, host range, restriction patterns, genome size and similarity of phage protein genes. All phages belonged to the Siphoviridae family. Five phages had prolate heads (52–61 × 40–52 nm) and 85–120 nm long non‐contractile tails (morphotype B2). Eight phages had small isometric heads (46–63 nm in diameter) and long non‐contractile tails (morphotype B1). The phages with the prolate heads showed a broader host range than the small isometric‐headed phages. The genome sizes of prolate phages were estimated between 15.9 and 16.3 kb while the genome sizes of small isometric‐headed phages were between 20.0 and 39.9 kb. Based on DNA restriction patterns, genome sizes and multiplex PCR analysis prolate phages were classified into the c2 group and were similar to the phage type c6A while all of the phages with small isometric heads were found to be related to the phage species 936.     

Filtration of milk fat globule membrane fragments from acid buttermilk cheese whey

The proteins and polar lipids present in milk fat globule membrane (MFGM) fragments are gaining attention for their technological and nutritional properties. These MFGM fragments are preferentially enriched in side streams of the dairy industry, like butter serum, buttermilk, and whey. The objective of this study was to recover MFGM fragments from whey by tangential filtration techniques. Acid buttermilk cheese whey was chosen as a source for purification by tangential membrane filtration because it is relatively rich in MFGM-fragments and because casein micelles are absent. Polyethersulfone and cellulose acetate membranes of different pore sizes were evaluated on polar lipid and MFGM-protein retention upon filtration at 40°C. All fractions were analyzed for dry matter, ash, lipids, proteins, reducing sugars, polar lipid content by HPLC, and for the presence of MFGM proteins by sodium dodecyl sulfate-PAGE. A fouling coefficient was calculated. It was found that a thermocalcic aggregation whey pretreatment was very effective in the clarification of the whey, but resulted in low permeate fluxes and high retention of ash and whey proteins. By means of an experimental design, the influence of pH and temperature on the fouling and the retention of polar lipids (and thus MFGM fragments), proteins, and total lipids upon microfiltration with 0.15 μM cellulose acetate membrane was investigated. All models were highly significant, and no outliers were observed. By increasing the pH from 4.6 to 7.5, polar lipid retention at 50°C increased from 64 to 98%, whereas fouling of the filtration membrane was minimized. A 3-step diafiltration of acid whey under these conditions resulted in a polar lipid concentration of 6.79 g/100 g of dry matter. As such, this study shows that tangential filtration techniques are suited for the purification of MFGM fragments.     

Biodiversity of lactococcal bacteriophages isolated from 3 Gouda-type cheese-producing plants

This study reports on the identification and characterization of bacteriophages isolated from cheese-production facilities that use undefined, mixed starter cultures. Phage screening was carried out on whey samples isolated from 3 factories, 2 utilizing one particular undefined starter mixture and 1 utilizing another undefined starter mixture. Phage screening was carried out using 40 strains isolated from the 2 mixed starter cultures, and phages were profiled using host range, electron microscopy, multiplex PCR, and DNA restriction analysis. Twenty distinct lactococcal phages were identified based on host range and DNA restriction profiles, all belonging to the 936-type phage species. Nineteen of these phages were found to be able to infect both recognized subspecies of Lactococcus lactis. Restriction of phage DNA isolated using a newly developed guanidinium thiocyanate disruption method showed that the genomes of the 20 isolated phages were between 26 and 31 kb in size. It is evident from this study that the use of mixed starters creates an ideal environment for the proliferation of different phages with slightly varying host ranges. Furthermore, in this environment, members of the 936-type phage species clearly dominated the phage population.     

Analysis of the complete genome sequence of the lactococcal bacteriophage bIBB29

Bacteriophage bIBB29 was isolated from a whey sample originating from an industrial biotechnological process, disturbed by a bacteriophage attack. Phage bIBB29 was determined to be active against three phage-resistant strains of Lactococcus lactis. It belongs to the 936 species containing virulent phages with isometric head and short non-contractile tail. One-step growth kinetics of bIBB29 phage showed that its latent time was 23 min, and the burst size was about 130 bacteriophages. The complete nucleotide sequence of the virulent L. lactis bacteriophage bIBB29 comprises 29305 nucleotides and is the sixth phage genome of the 936 species published until now. The G+C content of the bIBB29 genome (34.7%) is similar to that of its host and also to that of other phages from the 936 species. The bIBB29 genome counts 54 open reading frames organized in three typical clusters, corresponding to the early, middle and late expressed genes. Only 20 protein products of the predicted genes were found to have their homologs among proteins with known function. The early expressed region in the genomes of 936 group members displays the highest divergence, whereas the late and middle regions share high similarities, with the exception of five genes. The genome of bIBB29 shares the highest overall nucleotide similarity with bIL170 (87%), and the lowest with phage 712 (77%). The host range analysis showed that despite the high level of similarity between the receptor binding protein (RBP) of phage bIBB29 and P475, they have a different host range. This implies that RBP is not a sufficient factor for host range.     

Partition of lactic streptococcal bacteriophage during the ultrafiltration concentration of milk and whey

Milk and whey inoculated with lactic streptococcal bacteriophages 316, or 322, or both were concentrated by UF using a DDS Mini-Lab 20. The plate and frame unit was fitted with Type GR61PP polysulfone membrane with a 20,000 molecular weight cutoff. The unit was operated at an inlet pressure of .40 MPa and an outlet pressure of .23 MPa with an initial flux of 2.0 to 3.0 L/h. Samples of retentate, permeate, and membrane were analyzed for the presence of bacteriophages. Under the conditions established in this study, phage particles did not pass through the membrane but instead became trapped in the polarization concentration layer or in the membrane. Phages were recovered from the membrane by extraction in sterile buffered water with the Stomacher. The UF concentration of milk containing the host species of Streptococcus cremoris resulted in phage propagation and lysis of the host but did not result in the passage of phages through the membrane. The UF processing of milk or whey should produce a phage-free permeate.     

Lactococcal 936-species phage attachment to surface of Lactococcus lactis

The interactions of the 936-species phages sk1, jj50, and 64 with the cell surface of Lactococcus lactis LM0230 were analyzed. Cell envelopes (walls + plasma membrane), cell wall, or plasma membrane from L. lactis ssp. lactis LM0230 each inactivated the phages in vitro. However, other 936-species phages kh and P008, which do not infect strain LM0230, were not inactivated by any of the subcellular fractions. Treating cell walls or plasma membrane with the cell wall hydrolase mutanolysin eliminated inactivation of phage sk1. This suggested that intact cell wall fragments were required for inactivation. A role for plasma membrane in phage sk1 inactivation was further investigated. Boiling, washing in 2 M KCl, 8 M urea, or 0.1 M Na(2)CO(3)/pH 11, or treating the plasma membrane with proteases did not reduce adsorption or inactivation of phage. Adding lipoteichoic acid or antibodies to lipoteichoic acid did not reduce inactivation of phage in a mixture with membrane, suggesting that lipoteichoic acid was not involved. Inactivation by envelopes or cell wall correlated with ejection of DNA from the phage sk1 capsid. Although calcium is required for plaque formation, it was not required for adsorption, inactivation, or ejection of phage DNA by envelopes or cell wall. The results suggest that at least for phages sk1, jj50, and 64, adsorption and phage DNA injection into the host does not require a host membrane protein or lipoteichoic acid, and that cell wall components are sufficient for these initial steps of phage infection.     

Diversity of Streptococcus thermophilus phages in a large-production cheese factory in Argentina

Phage infections still represent a serious risk to the dairy industry, in which Streptococcus thermophilus is used in starter cultures for the manufacture of yogurt and cheese. The goal of the present study was to analyze the biodiversity of the virulent S. thermophilus phage population in one Argentinean cheese plant. Ten distinct S. thermophilus phages were isolated from cheese whey samples collected in a 2-mo survey. They were then characterized by their morphology, host range, and restriction patterns. These phages were also classified within the 2 main groups of S. thermophilus phages (cos- and pac-type) using a newly adapted multiplex PCR method. Six phages were classified as cos-type phages, whereas the 4 others belonged to the pac-type group. This study illustrates the phage diversity that can be found in one factory that rotates several cultures of S. thermophilus. Limiting the number of starter cultures is likely to reduce phage biodiversity within a fermentation facility.     

Effect of Whey Pretreatment on Composition and Functional Properties of Whey Protein Concentrate

The effect of pretreatment upon the composition and physicochemical and functional properties of whey, ultrafiltration (UF) retentate and freeze-dried and spray-dried whey protein concentrates (WPC) was investigated. Pretreatment was by cooling cheese whey to 0-5°C, adding calcium chloride, adjusting to pH 7.3, warming to 50°C, and removing the insoluble precipitate that formed by centrifugation or decantation. UF permeation flux rate of pretreated whey was about double that for control whey. Pretreated whey was essentially turbidity free, contained 85% less milkfat, 37% more calcium and 40% less phosphorus than whey. Pretreated whey WPC proteins were slightly more soluble at pH 3, but less functional for emulsification than whey WPC proteins. Neither whey WPC proteins nor pretreated whey WPC proteins was functional for foaming at 6% protein concentration.     

Gelation of casein micelles in ��-casein reduced milk prepared using membrane filtration

Pasteurized skim milk was subjected to membrane filtration using a molecular weight cut-off of 80 kDa and a plate and frame pilot scale system at temperatures below 10 °C. Via this process, transmission of whey proteins and ??-casein through the membrane was achieved. The milk was concentrated to two times (based on volume reduction), and whey protein-free permeate was added to return to the original volume fraction of casein micelles in milk. This diafiltration process was carried out four times, and the retentate obtained was nearly free of whey proteins and with approximately 20% of ??-casein removed. The same membrane filtration was also carried out at 25 °C to achieve transmission of whey protein but not of ??-casein, and to obtain whey protein-depleted milk without depletion of ??-casein.The gelling behaviour of these samples, reconstituted to the original casein volume fraction, was examined using rheology and diffusing wave spectroscopy. When compared to the original skim milk it was found that there were no statistically significant differences in gelation behaviour during acidification, but differences were noted in gelation time and final stiffness modulus for samples undergoing renneting. These differences were attributed mostly to the changes in ionic composition, as when the serum composition of the retentates was re-equilibrated against the original skim milk by dialysis; the gelation behaviour of the samples was comparable to that of skim milk. The results clearly indicate the importance of the milk's overall ionic balance in the early stages of aggregation of rennet-induced gelation of milk.     

Microfiltration Affinity Purification of Lactoferrin and Immunoglobulin G from Cheese Whey

A process combining affinity chromatography with membrane filtration was used to isolate lactoferrin and immunoglobulin G from Cheddar cheese whey. Heparin Sepharose, protein G Sepharose, and protein G bearing Streptococcal cells were used as adsorbents to form affinity complexes with target proteins. These could be retained by 0.2 μm microfiltration membranes and separated from unbound whey proteins. Binding capacities and binding constants of adsorbents determined by Langmuir-type adsorption isotherms were 124 mg/mL gel and 5.4 × 10^?6M for heparin Sepharose, 17.8 mg/mL gel and 3.5 × 10^?7M for protein G Sepharose, and 148 μ.g/mL cell suspension and 1.1 × 10^?7M for protein G cells. Lactoferrin with 95% purity and 92% iron-binding capacity, and immunoglobulin G with 90% purity and 86% activity could be obtained with reasonable yields.     

基于陶瓷膜技术的羊乳酪蛋白和其他组分的高效分级分离

为量化0.05 μm陶瓷膜脱除羊乳中乳清蛋白、乳糖、灰分、钙和磷的能力,在50 ℃条件下,脱脂乳进行3 倍浓缩,之后2 次间歇补水至原体积进行清洗过滤,最终得到1 份截留液、3 份透过液,并计算各组分总脱除率。结果表明：乳清蛋白脱除率为96.17%,乳糖脱除率为86.42%,灰分脱除率为73.39%,钙脱除率为34.90%,磷脱除率为55%。稀释过滤完毕后膜的纯水膜通量衰减系数为55.57%,使用质量分数为2%氢氧化钠和1%的硝酸溶液进行清洗,膜通量的恢复系数为99.21%。0.05 μm陶瓷膜可以实现羊乳酪蛋白和其他组分的有效分离,该技术适合在没有干酪乳清的条件下,以生鲜乳为原料加工酪蛋白胶束粉、乳清蛋白粉、乳糖等乳基配料产品。     

Detection and identification of Lactobacillus helveticus bacteriophages by PCR

A PCR protocol for detection of Lactobacillus helveticus bacteriophages was optimized. PCR was designed taking into account the sequence of the lys gene of temperate bacteriophage Phi-0303 and optimized to obtain a fragment of 222 bp using different Lb. helveticus phages from our collection. PCR was applied to total phage DNA extracted from 53 natural whey starters used for the production of Grana cheese and all gave the expected fragment. The presence of actively growing phages in the cultures was verified by traditional tests. Several PCR products of the lys gene were sequenced and aligned. The resulting sequences showed variable heterogeneity between the phages.     

Sieving effect of heat-denatured milk proteins during ultrafiltration of skim milk. I. The preliminary approach

The effect of heat treatment of skim milk on the ultrafiltration process was examined. The change in permeate collection rate was explained as a function of heat-induced modifications of the milk protein system. It is suggested that there was a sieving effect which contributed to the acceleration of permeate flow-down during membrane filtration. It is thought that this resulted from formation of complex structures between heat-denatured whey proteins and casein micelles.     

Use of ultrafiltration and supercritical fluid extraction to obtain a whey buttermilk powder enriched in milk fat globule membrane phospholipids

Whey buttermilk, a by-product from whey cream processing to butter, is rich in milk fat globule membrane (MFGM) constituents, which have technological and potential health properties. The objective of this work was to produce a dairy ingredient enriched in MFGM material, especially phospholipids, from whey buttermilk. Whey buttermilk was concentrated by ultrafiltration (10×) and subsequently diafiltered (5×) (10 kDa molecular mass cutoff membrane) at 25 °C and the final retentate was spray-dried. The whey buttermilk powder was submitted to supercritical extraction (350 bar, 50 °C) using carbon dioxide. The membrane filtration removed most of the lactose and ash from the whey buttermilk, and the supercritical extraction extracted exclusively non-polar lipids. The final powder contained 73% protein and 21% lipids, of which 61% were phospholipids. This ingredient, a phospholipids-rich dairy powder, could be used as an emulsifier in different food systems.