Manufacture of acid gels from skim milk using high-pressure homogenization

The effect of high-pressure homogenization (HPH) alone or in combination with a thermal treatment (TT) was investigated for the manufacture of acid gels from skim milk. Raw skim milk was subjected to HPH (0 to 350 MPa) or a TT (90°C, 5 min), or both, in the following processing combinations: 1) HPH, 2) HPH followed by TT, 3) TT followed by HPH, 4) TT, and 5) raw milk (control). After treatments, L* (lightness) values were measured, and then skim milk was acidified with 3% glucono-δ-lactone and rheological properties (G′ and gelation time), and whey holding capacity was evaluated. Treatments in which HPH and TT were combined showed greater L* values than those in which just HPH was applied. In all treatments, the L* values decreased as the pressure was increased up to 300 MPa with little change afterward. Gelation times were lower when HPH was combined with TT compared with the acid skim milk gels that were just pressure treated. The final G′ in gels obtained from skim milk subjected to the combined process (HPH and TT) was greater and pressure-dependent compared with all other gels. A maximum G′ (∼320 Pa) was observed with skim milk subjected to a combination of thermal processing before or after HPH at 350 MPa. Acid gels obtained from HPH milk at 350 MPa showed a linear decrease in whey holding capacity over time, retaining 20% more whey after centrifugation for 25 min compared with samples treated at lower pressures and all other treatments. Our results suggest that HPH in combination with TT can be used to improve the rheological properties and stability of yogurt, thus decreasing the need for additives.     

Quality of mango nectar processed by high-pressure homogenization with optimized heat treatment

This work aimed to evaluate the effect of high-pressure homogenization (HPH) with heat shock on Aspergillus niger, vitamin C, and color of mango nectar. The nectar was processed at 200 MPa followed by heat shock, which was optimized by response surface methodology by using mango nectar ratio (45 to 70), heat time (10 to 20), and temperature (60 to 85 °C) as variables. The color of mango nectar and vitamin C retention were evaluated at the optimized treatments, that is, 200 MPa + 61.5 °C/20 min or 73.5 °C/10 min. The mathematical model indicates that heat shock time and temperature showed a positive effect in the mould inactivation, whereas increasing ratio resulted in a protective effect on A. niger. The optimized treatments did not increase the retention of vitamin C, but had positive effect for the nectar color, in particular for samples treated at 200 MPa + 61.5 °C/20 min. PRACTICAL APPLICATION: The results obtained in this study show that the conidia can be inactivated by applying HPH with heat shock, particularly to apply HPH as an option to pasteurize fruit nectar for industries.     

High pressure homogenisation of raw whole bovine milk (a) effects on fat globule size and other properties

Although widely adopted by the chemical and pharmaceutical industries in recent years, little published data is available regarding possible applications of high pressure homogenisation for dairy products. The objective of this work was to compare the effects of conventional (18 MPa, two-stage) and single or two-stage high pressure homogenisation (HPH) at 50-200 MPa on some properties of raw whole bovine milk (approximately 4% fat). Fat globule size decreased as HPH pressure increased and, under certain conditions of temperature and pressure, HPH yielded significantly smaller fat globules than conventional homogenisation. Fat globule size was also affected by milk inlet temperature. The pH of all homogenised milk samples decreased during 24 h refrigerated storage. Total bacterial counts of milk were decreased significantly (P < 0.05) for milk samples HPH-treated at 150 or 200 MPa. Whiteness and rennet coagulation properties of milk were unaffected or enhanced, respectively, as homogenisation pressure was increased. Average casein micelle size decreased slightly when skim milk was homogenised at 200 MPa. Thus, HPH treatment has several, potentially significant, effects on milk properties.     

Factors affecting the inactivation of the natural microbiota of milk processed by pulsed electric fields and cross-flow microfiltration

Prior to processing milk and cream were standardised and homogenised. Skim milk was cross-flow microfiltered (CFMF) prior to treatment with pulsed electric fields (PEF) or high temperature short time (HTST) pasteurization. The effect of temperature of the skim milk and product composition on the efficacy of PEF treatment was determined. The electrical conductivity of the product was related to fat and solids content and increased 5% for every g/kg increase of solids and decreased by nearly 0·7% for every g/kg increase of fat. From the three microbial groups analyzed (mesophilic, coliform, and psychrotroph) in milks differences (P<0·05) in the inactivation of mesophilic microorganisms were observed between the counts following PEF treatment, while HTST pasteurization resulted in higher reductions in all different counts than those obtained after PEF. Increasing the skim milk temperature prior to PEF treatment to about 34°C showed equivalent reductions in microbial counts to skim milk treated at 6°C in half the time. The reductions achieved by a combination of CFMF and PEF treatments were comparable to those achieved when CFMF was combined with HTST pasteurization. A higher reduction in coliform counts was observed in homogenised products subjected to PEF than in products that were only standardised for fat content.     

Inactivation of Escherichia coli in milk by high-hydrostatic-pressure treatment in combination with antimicrobial peptides

We studied the inactivation in milk of four Escherichia coli strains (MG1655 and three pressure-resistant mutants isolated from MG1655) by high hydrostatic pressure, alone or in combination with the natural antimicrobial peptides lysozyme and nisin and at different temperatures (10 to 50 degrees C). Compared with that of phosphate buffer, the complex physicochemical environment of milk exerted a strong protective effect on E. coli MG1655 against high-hydrostatic-pressure inactivation, reducing inactivation from 7 logs at 400 MPa to only 3 logs at 700 MPa in 15 min at 20 degrees C. An increase in lethality was achieved by addition of high concentrations of lysozyme (400 microg/ml) and nisin (400 IU/ml) to the milk before pressure treatment. The additional reduction amounted maximally to 3 logs in skim milk at 550 MPa but was strain dependent and significantly reduced in 1.55% fat and whole milk. An increase of the process temperature to 50 degrees C also enhanced inactivation, particularly for the parental strain, but even in the presence of lysozyme and nisin, a 15-min treatment at 550 MPa and 50 degrees C in skim milk allowed decimal reductions of only 4.5 to 6.9 for the pressure-resistant mutants. A substantial improvement of inactivation efficiency at ambient temperature was achieved by application of consecutive, short pressure treatments interrupted by brief decompressions. Interestingly, this pulsed-pressure treatment enhanced the sensitivity of the cells not only to high pressure but also to the action of lysozyme and nisin.     

Potential applications of high pressure homogenisation in processing of liquid milk

Studies of the potential of high pressure homogenisation (HPH) for the combined pasteurisation/ homogenisation of raw bovine milk were undertaken. Raw milk was preheated to 45 degrees C and HPH-treated at 150, 200 or 250 MPa; milk outlet temperature at these pressures were 67, 76.8 and 83.6 degrees C, respectively, with a holding time of approximately 20 s. Raw and commercially pasteurized and homogenized (CPH) milk samples were analysed as controls. Fat globules in HPH samples were approximately half the size of those in CPH samples, although differences were not significant (P>0.05). beta-Lactoglobulin was denatured at pressures > or =150MPa, although little denaturation of alpha-lactalbumin was observed. Numbers of psychrotrophic bacteria in raw milk were reduced by 2.73 log cycles by HPH at 150 MPa and were uncountable following HPH at 200 or 250 MPa. Mesophilic bacterial counts were reduced by 1.30, 1.83 and 3.06 log cycles by HPH at 150, 200 or 250 MPa, respectively. No viable Staphylococcus aureus nor coliform cells remained in any HPH milk samples. HPH did not affect the colour of milk and HPH samples did not cream during refrigerated storage. The activities of plasmin, alkaline phosphatase and lactoperoxidase in milk were all greatly reduced by HPH. Pseudomonas fluorescens, inoculated into milk (approximately 10(6) cfu/ml), was reduced to undetectable levels by HPH at 200MPa (milk inlet temperature, approximately 10 degrees C); however, Ps. fluorescens proteinase was quite resistant to HPH under such conditions. Overall, owing to the significant increase in temperature and the possibility of varying the holding time, there may be potential applications for HPH as a novel liquid milk processing technique, combining many advantages of conventional homogenization and pasteurization of milk in a single process.     

Inactivation of Escherichia coli by high-pressure homogenisation is influenced by fluid viscosity but not by water activity and product composition

The inactivation of Escherichia coli MG1655 by high-pressure homogenisation (HPH) at pressures ranging from 100 to 300 MPa was studied in buffered suspensions adjusted to different relative viscosities (1.0, 1.3, 1.7, 2.7 and 4.9) with polyethylene glycol 6000 (PEG 6000). The water activity of these suspensions was not significantly affected by this high molecular weight solute. Bacterial inactivation was found to decrease with increasing viscosity of the suspensions, an effect that was more pronounced at higher pressures. To study the effect of water activity, series of E. coli suspensions having a different water activity (0.953-1.000) but the same relative viscosity (1.3, 1.7, 2.7 and 4.9) were made using PEG of different molecular weights (400, 600, 1000 and 6000), and subjected to HPH treatment. The results indicated that water activity does not influence inactivation. Finally, inactivation of E. coli MG1655 by HPH in skim milk, soy milk and strawberry-raspberry milk drink was found to be the same as in PEG containing buffer of the corresponding viscosity. These results identify fluid viscosity as a major environmental parameter affecting bacterial inactivation by HPH, as opposed to water activity and product composition, and should contribute to the development of HPH applications for the purpose of bacterial inactivation.     

Inhibitor‐Assisted High‐Pressure Inactivation of Bacteria in Skim Milk

The combined inactivation effects of high hydrostatic pressure (HHP) and antimicrobial compounds (potassium sorbate and ε‐polylysine [ε‐PL]) on 4 different bacterial strains present in skim milk and the effect of these treatments on milk quality were investigated in this study. HHP treatment at 500 MPa for 5 min reduced the populations of Escherichia coli, Salmonella enterica Typhimurium, Listeria monocytogenes, and Staphylococcus aureus from 6.5 log colony‐forming units (CFUs) or higher to less than 1 log CFU/mL. Compared to HHP alone, HHP with potassium or ε‐PL resulted in significantly higher reductions in the bacterial counts. After 5 min of treatment with HHP (500 MPa) and ε‐PL (2 mg/mL), no growth of E. coli, S. enterica Typhimurium, or L. monocytogenes in skim milk was observed during 15 d of refrigerated storage (4 ± 1 °C). Scanning electron microscopy analysis revealed that the synergistic treatments caused more serious damage to the bacterial cell walls. Quality assessments of the treated samples indicated that the combined treatments did not influence the color, the turbidity, the concentrations of –SH group of the proteins, or the in vitro digestion patterns of the milk. This study demonstrates that HHP with potassium or ε‐PL may be useful in the processing of milk or milk‐containing foods.     

Survival and growth of Yersinia enterocolitica strains inoculated in skimmed milk treated with high hydrostatic pressure

Four human pathogenic strains of Yersinia enterocolitica (serotypes O:1, O:3, O:8, and O:9) were inoculated (7-8 log CFU/ml) in UHT skimmed milk and treated at 300, 400, and 500 MPa for 10 min at 20 degrees C, and then kept at 8 degrees C to assess their evolution for 15 days. Treatments at 400 and 500 MPa caused the highest lethality, generally reaching counts below detection level (1 CFU/ml) in the culture media. At 300 MPa, the most baroresistant serotypes were O:3 and O:8. After 15 days of storage at 8 degrees C, Y. enterocolitica showed growth over 8 log (CFU/ml) in all treatments. Kinetic study of microbial inactivation in skimmed milk was performed with serotype O:8 at 300 MPa, showing a tailing after 35 min of pressure treatment.     

High pressure homogenisation of milk (b) effects on indigenous enzymatic activity

The objective of this study was to determine the effect of high pressure homogenisation (HPH) on alkaline phosphatase activity and plasmin and plasminogen-derived activities in raw whole bovine milk. Milk (approximately 4% fat) was treated by two-stage conventional homogenisation (18 MPa) or single or two-stage HPH at 50, 100, 150 or 200 MPa. Inactivation of plasmin and plasminogen-derived activities was evident in conventionally homogenised samples, and increased as HPH pressure increased. Two-stage HPH reduced both activities to a greater extent than single-stage HPH. Milk inlet temperature had a significant effect on residual plasmin and plasminogen activities of HPH-treated milk samples, especially those treated at 50 MPa. Inactivation of plasmin and plasminogen on HPH-treatment (150 MPa) of milk samples of varying fat contents (0-10%) was also investigated; there was a curvilinear relationship between residual plasmin and plasminogen-derived activities and fat content in the ranges 0-2% and 0-4%, respectively, with little additional inactivation at higher fat contents. Thus, indigenous proteolytic activity of milk is clearly affected by HPH. However, all homogenised milk samples retained active alkaline phosphatase, indicating that thermal conditions during HPH did not equate to that of conventional high temperature short time pasteurisation, and that the wide range of forces experienced by milk during HPH treatment does not inactivate the latter enzyme.     

Microbial shelf-life of high-pressure-homogenised milk

The anti-bacterial effect of high pressure homogenisation (HPH) on milk is widely reported but the shelf-life of HPH-treated milk, as reported in this communication has not been studied thus far. Raw whole milk was homogenised at 200 or 250 MPa at 55 or 70 °C and counts of total bacteria (TBC), psychrotrophs, pseudomonads, coliforms, lactobacilli, Bacillus cereus and Staphylococcus aureus were determined throughout subsequent storage for 14 days at 4 °C. Immediately after HPH treatment, counts of all bacteria were below the level of detection but after storage for 14 days at 4 °C, TBC, psychrotroph and pseudomonad counts had reached ∼10⁸ cfu mL⁻¹ in all samples treated with HPH. The limited shelf-life obtained indicates that HPH of milk at these processing parameters it is not a suitable alternative to pasteurisation for extending the shelf-life of milk.     

High pressure inactivation of Escherichia coli, Campylobacter jejuni, and spoilage microbiota on poultry meat

This study evaluated the high pressure inactivation of Campylobacter jejuni, Escherichia coli, and poultry meat spoilage organisms. All treatments were performed in aseptically prepared minced poultry meat. Treatment of 19 strains of C. jejuni at 300 MPa and 30°C revealed a large variation of pressure resistance. The recovery of pressure-induced sublethally injured C. jejuni depended on the availability of iron. The addition of iron content to enumeration media was required for resuscitation of sublethally injured cells. Survival of C. jejuni during storage of refrigerated poultry meat was analyzed in fresh and pressuretreated poultry meat, and in the presence or absence of spoilage microbiota. The presence of spoilage microbiota did not significantly influence the survival of C. jejuni. Pressure treatment at 400 MPa and 40°C reduced cell counts of Brochothrix thermosphacta, Carnobacterium divergens, C. jejuni, and Pseudomonas fluorescens to levels below the detection limit. Cell counts of E. coli AW1.7, however, were reduced by only 3.5 log (CFU/g) and remained stable during subsequent refrigerated storage. The resistance to treatment at 600 MPa and 40°C of E. coli AW1.7 was compared with Salmonella enterica, Shiga toxin-producing E. coli and nonpathogenic E. coli strains, and Staphylococcus spp. Cell counts of all organisms except E. coli AW 1.7 were reduced by more than 6 log CFU/g. Cell counts of E. coli AW1.7 were reduced by 4.5 log CFU/g only. Moreover, the ability of E. coli AW1.7 to resist pressure was comparable to the pressure-resistant mutant E. coli LMM1030. Our results indicate that preservation of fresh meat requires a combination of high pressure with high temperature (40 to 60°C) or other antimicrobial hurdles.     

Acid coagulation properties and suitability for yogurt production of cows’ milk treated by high-pressure homogenisation

The effects of high-pressure homogenisation (HPH) of cows’ milk were investigated for suitability for yogurt manufacture, compared with the processes currently applied in industry. Milk at different inlet temperatures (30 °C or 40 °C) was subjected to HPH treatment at 100, 200 or 300 MPa (one stage) and 130, 230 or 330 MPa (two-stage). HPH-treated milk was compared with milk heat-treated (90 °C for 90 s) and homogenised at 15 MPa, and with milk treated under the same thermal conditions and also fortified with 3% skim milk powder. Milk treated at 300 or 200 MPa showed higher gel strengths on coagulation, higher gel firmness in texture analysis, less syneresis and lower titratable acidity compared with conventionally treated milk fortified with 3% skim milk powder.     

Instant infusion pasteurisation of bovine milk. I. Effects on bacterial inactivation and physical–chemical properties

Two types of milk, skim milk and non-standardised raw milk, were heat treated using direct heating by instant infusion pasteurisation with treatment temperatures in the range from 72°C to 120°C and with holding times of less than 1 second. Indirect heating by HTST pasteurisation (72°C for 15 seconds) was used for comparison. The inactivation of microorganisms reached at least the same level when using instant infusion pasteurisation compared to HTST pasteurisation. Changes in the physical-chemical properties were observed in the skim milk fractions of instant infusion pasteurised non-standardised milk, whereas for instant infusion pasteurised skim milk less influence from the treatments was observed.     

Inactivation of spores of Bacillus cereus in cheese by high hydrostatic pressure with the addition of nisin or lysozyme

The objective of this work was to study high hydrostatic pressure (HHP) inactivation of spores of Bacillus cereus ATCC 9139 inoculated in model cheeses made of raw milk, together with the effects of the addition of nisin or lysozyme. The concentration of spores in model cheeses was approximately 6-log10 cfu/g of cheese. Cheeses were vacuum packed and stored at 8 degrees C. All samples except controls were submitted to a germination cycle of 60 MPa at 30 degrees C for 210 min, to a vegetative cells destruction cycle of 300 or 400 MPa at 30 degrees C for 15 min, or to both treatments. Bacillus cereus counts were measured 24 h and 15 d after HHP treatment. The combination of both cycles improved the efficiency of the whole treatment. When the second pressure-cycle was of 400 MPa, the highest inactivation (2.4 +/- 0.1 log10 cfu/g) was obtained with the presence of nisin (1.56 mg/L of milk), whereas lysozyme (22.4 mg/L of milk) did not increase sensitivity of the spores to HHP. For nisin (0.05 and 1.56 mg/L of milk), no significant differences were found between counts at 24 h and 15 d after treatment. Considering that mesophilic spore counts usually range from 2.6 to 3.0 log10 cfu/ml in raw milk, HHP at mild temperatures with the addition of nisin may be useful for improving safety and preservation of soft curd cheeses made from raw milk.     

Effects of the high pressure homogenization on the viability of yeast cell and volatile components in non-pasteurized rice wine

This study investigated the effects of high pressure homogenization (HPH) to replace a thermal pasteurization on the yeast cell inactivation and the volatile components of rice wine (RW). By applying HPH (172 MPa with 5 passes), the yeast count was reduced over 4 log cycles. The inactivation of yeast cells by HPH and a thermal processing, i.e. Holder pasteurization (HP), was quantitatively analyzed. In terms of the inactivation of yeast cells, the HPH at 172 MPa with 5 passes was equivalent to the HP at 65°C for 3 min. In RW, 34 volatile components were identified, including 8 alcohols, 18 esters, 2 acids, 1 carbonyls, 3 hydrocarbons, and 2 miscellaneous. Fruity volatiles were found more in HPH samples while components having fatty and oily characters were more detected in HP treated samples.     

Inactivation of Aspergillus niger in Mango Nectar by High-Pressure Homogenization Combined with Heat Shock

ABSTRACT:  This research evaluated the inactivation of a heat-resistant  Aspergillus niger  conidia in mango nectar by high-pressure homogenization (HPH) combined with heat shock.  A.niger  were inoculated in mango nectar (10⁶ conidia mL⁻¹) and subjected to HPH (300 to 100 MPa) and heat shock (80 °C for 5 to 20 min) before or after HPH. Processes were evaluated according to number of decimal reductions reached by each isolated or combined process. Scanning electron microscopy was performed to observe conidia wall after pressure treatment. Pressures below 150 MPa did not inactivate  A. niger  while pressures of 200 and 300 MPa resulted in 2 and more than 6 log reductions, respectively. D_{80 °C} of  A. niger  was determined as 5.03 min. A heat shock of 80 °C/15 min, reaching 3 decimal conidia reductions, was applied before or after a 200 MPa pressure treatment to improve the decimal reduction to 5 log cycles. Results indicated that HPH inactivated  A. niger  in mango nectar at 300 MPa (>6.24 log cycles) and that, with pressure (200 MPa) combined with post heat shock, it was possible to obtain the same decimal reduction, showing a synergistic effect. On the other hand, pre heat shock associated with HPH resulted in an additive effect. The observation of  A. niger  conidia treated by HPH at 100 and 200 MPa by scanning electron microscopy indicated that HPH promoted intense cell wall damage, which can sensitize the conidia to post heat shock and possibly explain the synergistic effect observed.
Practical Application : The results obtained in this paper are relevant to elucidate the mechanism of conidia inactivation in order to develop the application of HPH as an alternative pasteurization process for the fruit nectar industry.     

Reduction of counts of Listeria monocytogenes in cheese by means of high hydrostatic pressure

Inactivation of Listeria monocytogenes (strains NCTC 11994 and Scott A) was evaluated in model cheeses submitted to 10 min HHP treatments of 300, 400 or 500 MPa at 5 or 20 degrees C. Counts were measured immediately after high hydrostatic pressure (HHP) treatment (day 1) and after 2, 15 and 30 days of storage at 8 degrees C. Both strains behaved significantly different after 400 and 500 MPa, being NCTC 11994 more sensitive. Scarce differences were found among final values at both HHP treatment temperatures. Initial reductions (log cfu/g) for 400 MPa at 20 degrees C were 2.9 +/- 0.2 for strain NCTC 11994 and 1.5 +/- 0.2 for Scott A. They reached after 30-day storage 5.3 +/- 0.2 and 4.6 +/- 0.4 log cfu/g for NCTC 11994 and Scott A, respectively. For 500 MPa treatments, day-1 reductions of both strains were around 5-log cfu/g, and counts fell below quantification limit after 30 days. Injured cells (around 0.8-log cfu/g) were mostly observed in 400 MPa treated samples on days 1 and 2. Starter cells suffered higher inactivation and injury. For 20 degrees C treatments, its final counts (log cfu/g) at 300, 400 and 500 MPa were: 8.5 +/- 0.2, 5.4 +/- 0.3 and 2.5 +/- 0.1, respectively. These figures evidence the HHP potential to improve safety of cheese products.     

Inactivation of Bacillus cereus spores in milk by mild pressure and heat treatments

The objective of this work was to study the germination and subsequent inactivation of Bacillus cereus spores in milk by mild hydrostatic pressure treatment. In an introductory experiment with strain LMG6910 treated at 40 degrees C for 30 min at 0, 100, 300 and 600 MPa, germination levels were 1.5 to 3 logs higher in milk than in 100 mM potassium phosphate buffer (pH 6.7). The effects of pressure and germination-inducing components present in the milk on spore germination were synergistic. More detailed experiments were conducted in milk at a range of pressures between 100 and 600 MPa at temperatures between 30 and 60 degrees C to identify treatments that allow a 6 log inactivation of B. cereus spores. The mildest treatment resulting in a 6 log germination was 30 min at 200 MPa/40 degrees C. Lower treatment pressures or temperatures resulted in considerably less germination, and higher pressures and temperatures further increased germination, but a small fraction of spores always remained ungerminated. Further, not all germinated spores were inactivated by the pressure treatment, even under the most severe conditions (600 MPa/60 degrees C). Two possible approaches to achieve a 6 log spore inactivation were identified, and validated in three additional B. cereus strains. The first is a single step treatment at 500 MPa/60 degrees C for 30 min, the second is a two-step treatment consisting of pressure treatment for 30 min at 200 MPa/45 degrees C to induce spore germination, followed by mild heat treatment at 60 degrees C for 10 min to kill the germinated spores. Reduction of the pressurization time to 15 min still allows a 5 log inactivation. These results illustrate the potential of high-pressure treatment to inactivate bacterial spores in minimally processed foods.     

Calcium-Aggregated Milk: a Potential New Option for Improving the Viability of Lactic Acid Bacteria Under Heat Stress

Heat-induced inactivation of viable cells restricts a wide range of application of spray drying in producing dried lactic acid bacteria (LAB) products. In the present study, an effective method to enhance the stability of LAB under heat stress has been identified. This was done by enhancing the heat stability of skim milk as the carrier. Here, skim milk was supplemented with 10 mM CaCl₂ and heated to 90 °C for 10 min to induce protein aggregation. Using this Ca-aggregated skim milk as carrier, the survival of five LAB strains tested was found two orders of magnitudes higher than that of an untreated milk after the heating at a rising temperature from about 25 to 70 °C within 45 s. Possible mechanisms of the protection were explored by comparing the residual viability, microstructure of the cell-contained milk, and changes of suspension particle sizes caused by heat treatment of the four carriers, i.e., untreated milk, Ca-added milk, heat-treated milk, and Ca-aggregated milk. Ca-aggregated milk induced the highest microbial heat stability among them, providing a thick and compact encapsulation around LAB cells before the heat treatment for inactivation. The viable cells could stay in a comparatively more stable extracellular environment. This work reveals potentially a new option for using milk protein aggregates as a protectant of microorganisms. A series of calcium-enriched probiotic products may be developed based on the described principles of the finding of the Ca-aggregated milk.