Food Biotechnology, 21:129–143, 2007 Copyright © Taylor & Francis Group, LLC ISSN: 0890-5436 print DOI: 10.1080/08905430701410530
The Use of Viable and Heat-shocked Lactobacillus helveticus DPC 4571 in Enzyme-Modified Cheese Production 1532-4249 0890-5436 LFBT Food Biotechnology, Biotechnology Vol. 21, No. 2, MAY 2007: pp. 0–0
EMCLee B.H. Production et al. Using Lactobacillus helveticus
B.H. Lee1,2, K.N. Kilcawley1, J.A. Hannon1, S.Y. Park2, M.G. Wilkinson3, and T.P. Beresford1 1
Moorepark Food Research Centre, Teagasc, Moorepark, Fermoy, Cork, Ireland Department of Food Science & Agricultural Chemistry, McGill University, Montreal, Quebec, Canada 3 Department of Life Science, University of Limerick, Limerick, Ireland 2
The use of viable or attenuated Lactobacillus helveticus DPC 4571 for use in enzymemodified cheese production was assessed. Optimal heat shocking conditions for attenuation of DPC 4571 were found to be 69°C for 25 sec. Enzyme-modified cheese was produced from an emulsion of pre-hydrolysed rennet curd, water, and butter fat. This substrate was heat-treated and inoculated with either an equivalent level of viable or attenuated cells of DPC 4571 and further incubated under controlled conditions. The heat-treated products produced using attenuated DPC 4571 had a preferred sensory character with strong cheesy savory notes, enhanced secondary proteolysis, and more key volatile flavor compounds than those produced with viable DPC 4571. However, prolonged incubation (>16 h) resulted in growth of advantageous enterococci, which adversely influenced the sensory profile. Key Words: enzyme-modified cheese; Lactobacillus helveticus DPC 4571; attenuation
INTRODUCTION Enzyme-modified cheeses (EMCs) are high-intensity cheese flavor ingredients widely used as an economical alternative to natural cheese in processed foods (Moskowitz and La Belle, 1981; Moskowitz and Noelck, 1987; Kilcawley et al., 1998). The increased demand for EMC in recent years is due to a greater
Address correspondence to B.H. Lee, Department of Food Science and Agricultural Chemistry, McGill University, 21, III Lakeshore Road, Ste-Anne-de-Bellevue, QC H9X 3V9/ Food R&D Centre, AAFC, Canada; E-mail:
[email protected].
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demand for convenience foods where EMCs are widely used as partial or whole replacement of natural cheese. EMCs are also used to reduce fat content or to replace fat functionality in low-fat products due to health-related concerns of saturated fat and cholesterol in traditional dairy products (Buhler, 1995). The range of EMC cheese products and applications are diverse, and they are generally added to foods at levels of 0.15–2.0% by weight with some replacing up to 50% of the natural cheese (Buhler, 1995). Thus EMCs are cost effective, nutritious, and natural cheese ingredients. Lactobacilli are a good source of peptidases, proteinases, and esterases. Consequently, and thus intensive research has been carried out using these bacteria to accelerate cheese ripening (Lee et al., 1990; Abboudi et al., 1991; Trépanier et al., 1992; Habibi-Najafi and Lee, 1994; Choi et al., 2004) and in EMC production (Park et al., 1995). However, a major problem associated with using lactobacilli as adjunct starter to cheese milk or in EMC production is rapid and excess acidification, resulting in atypical and low-quality cheese flavor. Heat-treatment of lactobacilli or lactococci inactivates lactose fermenting ability, without significant losses of peptidase and proteinase activity (Abboudi et al., 1991). The objective of this study was to investigate the potential of viable and heatshocked cells of Lactobacillus helveticus DPC 4571 in the production of EMC.
MATERIALS AND METHODS Preparation of Viable and Heat-Shocked Cells of L. helveticus DPC 4571 L. helveticus DPC 4571 was maintained at −70°C in 10% (w/v) in skim milk (Moorepark Food Research Centre, Teagasc, Moorepark, Fermoy, Cork, Ireland). This organism was revived by two consecutive inoculations and was grown for 16 h at 37°C in 200 mL or 2000 mL of MRS (Oxoid). Tubes containing maximum recovery diluent (pH 7.0; Oxoid) were pre-incubated at different temperatures (65°C, 67°C, 69°C, 71°C) for 5 min. The cells were heat-shocked by injecting 1.0 ml of the cell suspension held at 45°C to 9 ml of pre-incubated diluent held at either 65°C, 67°C, 69°C, 71°C. The mixture was agitated in a water bath (Heto Lab Equipment, Denmark) and held at a desired temperature (65°C, 67°C, 69°C, or 71°C) for 15 sec, 20 sec, 25 sec, or 30 sec, then rapidly cooled by immersion in ice. Viable counts of the cells and enzyme activities (x-prolyldipeptidyl peptidase and general aminopeptidase) before and after the heat treatment were evaluated. Portions (1 ml) of non-heat shocked cells (control) and heat-shocked cells were inoculated into 8 ml of 10% reconstituted skim milk (RSM) and incubated for 32 h at 37°C to investigate the retardation of lactic acid production by measuring pH at 16 and 32 h using a pH meter (PHM82, Copenhagen, Denmark).
EMC Production Using Lactobacillus helveticus
To determine intracellular enzyme activities, cell suspensions of the above were first centrifuged at 5,000 g for 10 min mixed with 0.8 g of 0.1 mm beads (Biospec Products, Milford, Il., USA) in 0.8 ml of 0.01 M sodium phosphate buffer (pH 7.0) and disrupted at 3 min intervals over 12 min in cold room by Mini Beadbeater™-8 (Biospec Products, Howard Ind., Milford, Il., USA). To study the effect of heat-shocking over time on acid production, selected heat-treated cell suspensions were inoculated into 10% (w/v) antibiotic free low-heat skim milk containing an indicator, 1% phenolphthaline, and incubated at 37°C. The pH was measured at 0, 16, and 32 h using a pH meter (PHM82, Copenhagen, Denmark).
Enzyme-Modified Cheese Preparation Batches of EMC (1 kg or 4 kg) were produced in sterile 2 L reactors (B.Braun, Melsungen, Germany) by incubating an emulsion of rennet curd, water, and anhydrous butter fat with two commercial enzymes, 0.42% Savorase RST100 (Danisco A/S, Copenhagen, Denmark) and 0.11% Lipomod 338 (Biocatalysts, Wales, UK) for 6 h at 45°C at 100 rpm agitation. The EMC was heat treated to inactivate the enzymes at 80°C for 30 min, and separated into two batches (0.5 kg or 2 Kg). Each batch was inoculated with either viable (1010 cfu/mL) or an equivalent level of heat-shocked cells, then further incubated at 37°C 100 rpm agitation for 24 h.
Microbiological Analysis EMC samples were evaluated for microbial content during ripening. Microbial evolution was measured as follows: total counts using Milk Plate Count Agar (MPCA, Merck), non-starter lactic acid bacteria (NSLAB) using LBS (Oxoid), enterococci using Kanamycin-Asulin-Azid (KAA, Merck) agar, thermophilic lactobacilli using MRS5.4 adjusted with acetic acid before sterilization, and total coliforms using violet red bile (VRB) agar. One mL of serial dilutions was added into duplicate petri dishes first and molten agar at 45°C was pour plated by swirling. The media and conditions used were: plate count agar incubated for 48 h at 30°C, KAA for 24 h at 37°C, VRB agars for 24 h at 37°C all aerobically, LBS for 48 h at 30°C, and MRS5.4 for 48 h at 37°C anaerobically.
Protein and Enzyme Assays Protein was estimated using the Bio-Rad protein assay (Bio-Rad Laboratories GmbH, Munich, Germany) using bovine serum albumin as standard. EMC samples were centrifuged twice (5,000 × g) for 10 min, and the supernatants were assayed directly for enzyme activities. X-prolyldipeptidyl peptidase (PepX) activity was measured using glycyl-l-prolyl AMC (aminomethyl coumarin)
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according to the method of Habibi-Najafi and Lee (1991), with some modifications. The AMC substrate was measured at the excitation and emission wavelengths of 370 and 440 nm, respectively, using a spectrofluorometer (Kontron Instument, SFM 25, Zurich, Switzerland). The amount of AMC released was calculated from a standard curve. Sample (50 μL) was added to 900 μL of 0.05M sodium phosphate (pH 7.5), and incubated at 37°C for 5 min, and the reaction was initiated by adding 50 μL of 1mM substrate. After incubation at 37°C for 20 min, the reaction was terminated by adding 1 mL of 1.5 M acetic acid. One unit of enzyme activity was defined as the amount of enzyme necessary to release 1 nM of AMC per min per mL. General aminopeptidase activity (PepN) was measured using lysyl-p-nitroanilide according to Arora and Lee (1994). PepN was measured in the mixture containing 100 μL of sample supernatant, 285 μL of 0.01 M sodium phosphate buffer (pH 7.0), and 50 μL of substrate (16.4 mM) incubated for 30 min at 37°C. An enzyme unit was defined as nM chromophore released per min per mL. Proteinase activity was measured in a mixture of 1.0 mL azocasein (11 mg/ml; Sigma) dissolved in 0.05M phosphate buffer (pH 7.0) and 100 μL of sample in the mixture of 50 μL of 16.4 mM (Park et al., 1995). After incubation for 30 min at 37°C, the reaction was terminated by adding 2 mL of 12% (w/v) TCA solution. The mixture was centrifuged at 10,000 g for 5 min, and the supernatant was used to measure the absorbance at 340 nm. One unit of enzyme activity was defined as the amount of enzyme necessary to increase 0.01-absorbance units per min at 340 nm (Park et al., 1995).
Composition EMC samples were analyzed for NaCl (IDF, 1979), fat (IDF, 1996), total nitrogen (IDF, 1986), and moisture by drying in a force draft oven at 110°C for 16 h (IDF, 1982). The pH of EMC was directly measured on a slurry (20 g) using an Orion Model 720A pH meter (Orion Res. Inc., Boston, Mass., USA).
Assessment of Proteolysis Proteolysis was estimated by measuring the percentage of total N soluble in water at pH 4.6 (pH4.6 WSN) and in 5% phosphotungstic acid (PTA), according to the method of Kuchroo and Fox (1982). Free amino acids (FAA) were determined on 12% trichloroacetic acid (TCA) filtrates prepared from the pH 4.6 water soluble fraction (Wilkinson et al., 1992). Filtrates were analyzed using a Beckman 6300 Analyzer (Beckman, High Wycombe, Bucks, UK) with results expressed as ug FAA per g of EMC.
Assessment of Acetic, Butyric and Caproic Acids Acetic, butyric, and caproic acids were recovered by steam distillation and subsequently determined by ligand-exchange, ion-exclusion HPLC, according
EMC Production Using Lactobacillus helveticus
to the method of Kilcawley et al. (2000), and results were expressed as ppm of sample.
Assessment of Volatiles Frozen EMC samples were thawed at room temperature (20°C). A 5 g portion was weighed into a 35 mL fritted glass sparger. The tube containing the sample was pre-heated to 37°C and purged for 15 min with 40 mL min−1 helium. The volatiles emitted under these conditions were swept onto a Tenax/ silica gel/charcoal Trap (Supelco, Sigma-Aldrich, Dublin, Ireland). Desorption and enrichment of trapped volatiles were carried out with a Techmar model 3000 Purge and Trap (Techmar Co., Cincinnati, Ohio, USA) as follows: the traps were preheated rapidly to 225°C, desorbed for 2 min, and injected into a DB-5 column (30 × 0.25 i.d., 1μm film thickness, J & W Scientific, Folsam, Calif., USA) using a Varian Star 3400 GC (JVA Analytical Ltd, Dublin, Ireland). The column was operated with helium carrier gas at a flow of 1 mL min−1. Initial injector temperature was 50°C, held for 2.1 min ramped to 200°C at 180°C min−1. Initial column temperature was −60°C, held for 3 min ramped to 30°C at 50°C min−1, held for 5 min, ramped to 60°C at 10°C min−1, ramped to 250°C at 20°C min−1 and held for 1 min. Total run time was 23.30 min. The Gas Chromatogram (GC) column was connected directly to the ion source of a Varian Saturn 2000 mass spectrometer (JVA Analytical). The mass analyzer was set at 35–350 m/z, ionisation voltage of 70eV, multiplier voltage 1340 V. Compounds were identified by comparing retention times with authentic compounds under identical operating conditions, running a series of n-alkanes and comparing Kovats retention indices with published literature and comparison of mass spectra with on-line Nist 98 Library of standard compounds. For quantification the average peak areas of duplicate runs were determined. Principal component analysis (PCA) was carried out on the mass spectra data in order to get the best possible analysis of the volatile flavor components.
Sensory Analysis Three panelists familiar with cheddar cheese were used to comment on three sets of five EMC samples (0, 16, 24, 40 & 48 h) on a 1 to 5 scale, with 5 best and 1 worst. Eight panelists familiar with cheddar cheese were used to rank triplicate EMC samples (0, 8, 16 & 24 h) for cheesy flavor using the method of Jellinek (1985). EMC samples (10 g) were coded with randomly selected 4-digit numbers and presented. Panelists were asked to rate the order of tasting between and within days to account for first order carry-over effects (Macfie et al., 1989).
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Statistical Analysis The data from microbial, chemical, enzymatic, and sensory analyses were evaluated for their significance (p
