Mark A.E. Auty

Comparative survival rates of human-derived probiotic Lactobacillus paracasei and L. salivarius strains during heat treatment and spray drying.

ABSTRACT Spray drying of skim milk was evaluated as a means of preservingLactobacillus paracasei NFBC 338 and Lactobacillus salivarius UCC 118, which are human-derived strains with probiotic potential. Our initial experiments revealed that NFBC 338 is considerably more heat resistant in 20% (wt/vol) skim milk than UCC 118 is; the comparable decimal reduction times were 11.1 and 1.1 min, respectively, at 59°C. An air outlet temperature of 80 to 85°C was optimal for spray drying; these conditions resulted in powders with moisture contents of 4.1 to 4.2% and viable counts of 3.2 × 109 CFU/g for NFBC 338 and 5.2 × 107 CFU/g for UCC 118. Thus, L. paracasei NFBC 338 survived better than L. salivarius UCC 118 during spray drying; similar results were obtained when we used confocal scanning laser microscopy and LIVE/DEAD BacLight viability staining. In addition, confocal scanning laser microscopy revealed that the probiotic lactobacilli were located primarily in the powder particles. Although both spray-dried cultures appeared to be stressed, as shown by increased sensitivity to NaCl, bacteriocin production by UCC 118 was not affected by the process, nor was the activity of the bacteriocin peptide. The level of survival of NFBC 338 remained constant at ∼1 × 109 CFU/g during 2 months of powder storage at 4°C, while a decline in the level of survival of approximately 1 log (from 7.2 × 107 to 9.5 × 106 CFU/g) was observed for UCC 118 stored under the same conditions. However, survival of both Lactobacillus strains during powder storage was inversely related to the storage temperature. Our data demonstrate that spray drying may be a cost-effective way to produce large quantities of some probiotic cultures.

Direct In Situ Viability Assessment of Bacteria in Probiotic Dairy Products Using Viability Staining in Conjunction with Confocal Scanning Laser Microscopy

ABSTRACT The viability of the human probiotic strains Lactobacillus paracasei NFBC 338 and Bifidobacterium sp. strain UCC 35612 in reconstituted skim milk was assessed by confocal scanning laser microscopy using the LIVE/DEAD BacLight viability stain. The technique was rapid (<30 min) and clearly differentiated live from heat-killed bacteria. The microscopic enumeration of various proportions of viable to heat-killed bacteria was then compared with conventional plating on nutrient agar. Direct microscopic enumeration of bacteria indicated that plate counting led to an underestimation of bacterial numbers, which was most likely related to clumping. Similarly, LIVE/DEAD BacLight staining yielded bacterial counts that were higher than cell numbers obtained by plate counting (CFU) in milk and fermented milk. These results indicate the value of the microscopic approach for rapid viability testing of such probiotic products. In contrast, the numbers obtained by direct microscopic counting for Cheddar cheese and spray-dried probiotic milk powder were lower than those obtained by plate counting. These results highlight the limitations of LIVE/DEAD BacLight staining and the need to optimize the technique for different strain-product combinations. The minimum detection limit for in situ viability staining in conjunction with confocal scanning laser microscopy enumeration was ∼108 bacteria/ml (equivalent to ∼107 CFU/ml), based on Bifidobacterium sp. strain UCC 35612 counts in maximum-recovery diluent.

Development and application of confocal scanning laser microscopy methods for studying the distribution of fat and protein in selected dairy products.

Confocal scanning laser microscopy (CSLM) methods were developed to identify fat and protein in cheeses milk chocolate and milk powders. Various fluorescent probes were assessed for their ability to label fat or protein in selected food products in situ. Dual labelling of fat and protein was made possible by using mixtures of probes. Selected probes and probe mixtures were then used to study (a) structure development of Mozzarella cheese during manufacture and ripening, and (b)) the distribution of fat and protein in milk chocolate made with milk powders containing varying levels of free fat. Microstructural changes in the protein and fat phases of Mozzarella cheese were observed at each major step in processing. Aggregation of renneted micelles occurred during curd formation; this was followed by amalgamation of the para-casein into linear fibres during plasticization. Following storage, the protein phase of the Mozzarella became more continuous; entrapping and isolating fat globules. Chocolate made with a high free-fat spray-dried powder blend showed a homogeneous fat distribution, similar to that of chocolate made with roller-dried milk. Chocolate made with whole milk powder containing 10 g free fat/100 fat showed a non-homogeneous fat distribution with some fat occluded within milk protein particles. These differences in fat distribution were related to Casson yield value and Casson viscosity of the chocolates.

Influence of sodium caseinate and whey protein on baking properties and rheology of frozen dough

ABSTRACT Dairy ingredients are added to bakery products to increase nutritional and functional properties. Sodium caseinate (SC) and whey protein concentrate (WPC) were incorporated into frozen dough. WPC was subjected to heat treatment (WPCHT) to eliminate undesirable weakening of the gluten network. 2% SC or 4% SC decreased proof time, increased loaf volume, and improved texture. Effects of adding 4% SC on baking quality were similar to adding ascorbic acid (AA) and diacetyl tartaric acid esters of monoglycerides (DATEM). WPC increased proof time, decreased volume, and negatively affected texture. Heat treatment of WPC improved baking performance. Bread with WPCHT had volume similar to that of the control without dairy ingredients. Adding 4% SC decreased resistance to extension (R5cm measured with the extensigraph), while adding 4% WPC increased extensibility. Dynamic oscillation testing determined the effects of the ingredients on fundamental rheological properties. WPC decreased storage modulus (G′) a...

Characterization of β-Lactoglobulin Fibrillar Assembly Using Atomic Force Microscopy, Polyacrylamide Gel Electrophoresis, and in Situ Fourier Transform Infrared Spectroscopy

The aggregation process of beta-lactoglobulin (beta-lg) from 0 min to 20 h was studied using atomic force microscopy (AFM), scanning transmission electron microscopy (STEM), sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and in situ attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR). Fibril assembly was monitored in real time using AFM up to 20 h. From 0 to 85 min, beta-lg monomers deformed and expanded with some aggregation. After 85 min, fibrillar structures were formed, exceeding 10 mum in length. Fibrillar structures were confirmed by STEM. Secondary structural changes occurring during fibril formation were monitored by ATR-FTIR at 80 degrees C and indicated a decrease in alpha-helix content and an increase in beta-sheet content. SDS-PAGE indicated that fibrils were composed of polypeptides and not intact monomers. In this study, beta-lg and whey protein isolate (WPI)-derived fibrils, including some double helices, in water were observed by AFM under ambient conditions and in their native aqueous environment.

The effect of high pressure treatment on the functional and rheological properties of Mozzarella cheese

Abstract Low-moisture Mozzarella cheese (LMMC) was high pressure (HP) treated at different stages of storage at 4°C and analysed immediately post HP treatment. Confocal laser scanning microscopy of the unheated cheese indicated that HP treatment enhanced the development of age-related swelling of the paracasein matrix. This microstructural change coincided with a reduction in the level of serum expressed on centrifugation and suggests that HP results in an increase in the water holding capacity of the paracasein matrix. Proteolysis, as measured by urea polyacrylamide gel electrophoresis (urea-PAGE), pH 4.6 water soluble nitrogen and total levels of free amino acids (FAA), was largely unaffected by HP treatment of LMMC. HP treatment resulted in an increase in the flowability and a reduction in the melt time on heating at 280°C, especially at storage times ≤15 days. Dynamic measurement of the viscoelastic changes on heating the cheese from 20 to 82°C showed that HP treatment resulted in an increase in the fluidity of the heated cheese, as measured by phase angle, especially in 1-day-old cheese. Thus, accelerated ripening of LMMC was induced by HP treatment, which may lead to the development of an industrial process if cost effective commercial HP equipment were available.

The manufacture of miniature Cheddar-type cheeses from milks with different fat globule size distributions

A novel 2-stage gravity separation scheme was developed for fractionation of raw, whole bovine milk into fractions enriched in small (SFG) or large (LFG) fat globules. The volume mean diameter of fat globules in SFG, LFG or control (CTRL) milk was 3.45, 4.68 and 3.58 microm, respectively. The maximum in storage modulus (index of firmness) decreased with increasing fat globule size for rennet-induced gels formed from SFG, LFG or CTRL milks. Miniature (20 g) Cheddar cheeses were manufactured using each of the 3 milks. There were no significant (P > 0.05) differences in the pH, moisture and fat in dry matter levels between cheeses made using any of the 3 milks, however, the fat content of the cheese made using SFG milk was approximately 1% lower than that of cheese made using LFG or CTRL milk in each of the 2 trials. Image analysis of confocal scanning laser micrographs of the cheeses illustrated that the star volume of fat globules in the cheeses decreased significantly (P < or = 0.05) as the size of fat globules in the milks used for cheesemaking was reduced. This indicates that it is possible to manipulate the size distribution of fat globules in Cheddar cheese by adjusting the fat globule size distribution of the milk used for cheese-making. The concentration of free fatty acids (FFA) increased in all cheeses during ripening. At 120 d of ripening, the concentration of FFA varied significantly (P < or = 0.05 and P < or = 0.001 for trials 1 and 2, respectively) with fat globule size, with cheeses made in trial 2 from LFG, SFG or CTRL milks having total FFA levels of 3391, 2820 and 2612 mg/kg cheese, respectively.

Effect of exopolysaccharide produced by isogenic strains of Lactococcus lactis on half-fat Cheddar cheese.

Fat-reduced cheeses often suffer from undesirable texture, flavor, and cooking properties. Exopolysaccharides (EPS) produced by starter strains have been proposed as a mechanism to increase yield and to improve the texture and cooking properties of reduced-fat cheeses. The objective of this work was to assess the influence of an exopolysaccharide on the yield, texture, cooking properties, and quality of half-fat Cheddar cheese. Two pilot-scale half-fat Cheddar cheeses were manufactured using single starters of an isogenic strain of Lactococcus lactis ssp. cremoris (DPC6532 and DPC6533) that differed in their ability to produce exopolysaccharide. Consequently, any differences detected between the cheeses were attributed to the presence of the exopolysaccharide. The results indicated that cheeses made with the exopolysaccharide-producing starter had an 8.17% increase in actual cheese yield (per 100 kg of milk), a 9.49% increase in moisture content, increase in water activity and water desorption rate at relative humidities <or=90%, significant differences in the cheeses microstructure, and a significant improvement in both textural and cooking properties, without negatively affecting the flavor profiles of the cheeses.

Effect of varying high-pressure treatment conditions on acceleration of ripening of cheddar cheese

The effects of high-pressure (HP) treatment conditions on proteolysis in Cheddar cheese ripening were investigated by response surface analysis. A second-order central composite rotatable design (CCRD) was used to study the influences of pressure and processing time in the range 70–400 MPa and 3.5–81.5 h, respectively, at 25 °C. Separate control samples were maintained for equivalent times at 25 or 8 °C at atmospheric pressure. Urea-PAGE analysis indicated that breakdown of αs1-casein, and concomitant production of αs1-I-casein (f 24–199), was increased by treatment at 100 MPa for 70 h at 25 °C. Accumulation of αs1-I-casein did not increase at >225 MPa and, at 350–400 MPa, decreased accumulation of αs1-I-casein was observed. Response surface models indicated that treatment at pressures <150 MPa gave greatest increases in levels of pH 4.6-soluble N (SN), expressed as a percentage of total N (TN) in cheese, relative to control samples. As pressurisation time increased at <150 MPa, levels of pH 4.6-SN/TN in cheese increased. However, in general, HP reduced the production of free amino acids. Confocal laser scanning microscopy showed structural differences between Cheddar cheese treated at 350–400 MPa and control cheese stored at 25 °C or 8 °C. In summary, the application of relatively low pressures increased levels of primary proteolysis in Cheddar cheese. However, the temperature of pressurisation was a significant determinant of the overall effect of HP on proteolysis in Cheddar cheese.

Growth and location of bacterial colonies within dairy foods using microscopy techniques: a review.

The growth, location, and distribution of bacterial colonies in dairy products are important factors for the ripening and flavor development of cheeses, yogurts, and soured creams. Starter, non-starter, spoilage, and pathogenic bacteria all become entrapped in the developing casein matrix of dairy foods. In order to visualize these bacterial colonies and the environments surrounding them, microscopy techniques are used. The use of various microscopy methods allow for the rapid detection, enumeration, and distribution of starter, non-starter and pathogenic bacteria in dairy foods. Confocal laser scanning microscopy is extensively utilized to identify bacteria location via the use of fluorescent dyes. Further study is needed in relation to the development of micro- gradients and localized ripening parameters in dairy products due to the location of bacteria at the protein–fat interface. Development in the area of bacterial discrimination using microscopy techniques and fluorescent dyes/tags is needed as the benefits of rapidly identifying spoilage/pathogenic bacteria early in product manufacture would be of huge benefit in relation to both safety and financial concerns.