S.A. Rankin

Invited review: The application of alkaline phosphatase assays for the validation of milk product pasteurization

Standard practices for indirectly assessing the pasteurization status of milk products are primarily based on the thermal inactivation kinetics of the endogenous milk enzyme, alkaline phosphatase (ALP). This assessment provides an invaluable, if not required, tool for both regulatory and in-house process control and validation. Endogenous milk ALP manifests a slightly higher heat resistance than the pathogenic microflora upon which pasteurization time and temperature requirements are based. Hence, ALP activity is recognized and accepted as the method of choice for the rapid validation of milk product pasteurization. However, ALP assays have notable limitations that must be understood if they are to be administered and interpreted correctly and the results are to be applied judiciously. Issues such as the reactivation of heat-denatured ALP and the presence of both heat-stable and -labile microbial ALP are addressed. A discussion of ALP in the milk of nonbovine species is presented based on the limited literature available. Some discussion of research involving alternative pasteurization indicators also is presented. This article is intended to summarize the pertinent details of the ALP assay for dairy products (noting the basis and limitations of various methods) and the processing, handling, and known compositional factors that influence the assay results.

Lactobacillus casei metabolic potential to utilize citrate as an energy source in ripening cheese: a bioinformatics approach

Aims:  To identify potential pathways for citrate catabolism by Lactobacillus casei under conditions similar to ripening cheese.

Purification and high-resolution top-down mass spectrometric characterization of human salivary α-amylase.

Human salivary α-amylase (HSAMY) is a major component of salivary secretions, possessing multiple important biological functions. Here we have established three methods to purify HSAMY in human saliva for comprehensive characterization of HSAMY by high-resolution top-down mass spectrometry (MS). Among the three purification methods, the affinity method based on the enzyme-substrate specific interaction between amylase and glycogen is preferred, providing the highest purity HSAMY with high reproducibility. Subsequently, we employed Fourier transform ion cyclotron resonance MS to analyze the purified HSAMY. The predominant form of α-amylase purified from saliva of various races and genders is nonglycosylated with the same molecular weight of 55,881.2, which is 1885.8 lower than the calculated value based on the DNA-predicted sequence. High-resolution MS revealed the truncation of the first 15 N-terminal amino acids (-1858.96) and the subsequent formation of pyroglutamic acid at the new N-terminus Gln (-17.03). More importantly, five disulfide bonds in HSAMY were identified (-10.08) and effectively localized by tandem MS in conjunction with complete and partial reduction by tris (2-carboxyethyl) phosphine. Overall, this study demonstrates that top-down MS combined with affinity purification and partial reduction is a powerful method for rapid purification and complete characterization of large proteins with complex and overlapping disulfide bond patterns.

Antimicrobial Properties of Commercial Annatto Extracts against Selected Pathogenic, Lactic Acid, and Spoilage Microorganisms

Annatto preparations are used to impart distinctive flavor and color to foods and are a primary colorant in dairy foods such as cheese and butter. There are several reports indicating that certain fractions of the annatto plant have biological activities against microorganisms of significance in food fermentation, food preservation, and human health. However, little is reported describing the nature of the antimicrobial compound(s) or their potential presence in commercial annatto colorant preparations. This study was conducted to determine whether commonly available annatto extracts are capable of influencing the outgrowth of selected lactic acid, spoilage, and pathogenic microorganisms. Disk diffusion and tube macrodilution techniques were used to determine the MICs and MBCs of double-strength water-soluble annatto extracts. Standard antibiotic disks were used as controls for the disk diffusion assay. The results demonstrate that annatto has an inhibitory effect on Bacillus cereus, Clostridium perfringens, and Staphylococcus aureus, with MICs of 0.08, 0.31, and 0.16% (vol/vol) and diameters of inhibition of 9 to 10, 12 to 13, and 15 to 16 mm, respectively. A concentration of 0.63% (vol/vol) inhibited the growth of Streptococcus thermophilus, Lactobacillus casei subsp. casei, Lactococcus lactis, and Paenibacillus polymyxa. The MICs for Listeria monocytogenes and Enterococcus durans were 1.25 and 2.5% (vol/vol), respectively. No activity was detected against Lactobacillus plantarum, Bifidobacterium bifidum, yeasts, or selected gram-negative bacteria.

Determining the impact of varying levels of cherry powder and starter culture on quality and sensory attributes of indirectly cured, emulsified cooked sausages

Indirect curing is a process that utilizes ingredients high in naturally occurring nitrate and a nitrate reducing bacterial starter culture (SC) to provide quality and sensory attributes similar to nitrite-added cured meats. The objective of this study was to determine the effects varying concentrations of starter culture and the addition of cherry powder (CP) had on improving quality and sensory attributes of indirectly cured sausages. Four treatments (TRTs) (TRT 1: low SC+no CP; TRT 2: low SC+CP; TRT 3: high SC+no CP; and TRT 4: high SC+CP) and a sodium nitrite-added (156 ppm) control were investigated. Residual nitrite levels throughout storage declined most rapidly in TRTs 2 and 4 (P<0.05). Few differences existed between TRTs and C for pH, objective color, or cured pigment concentrations. Consumer sensory panel scores revealed all treatment combinations were comparable (P>0.05) to the C for all sensory attributes.

Growth of Lactobacillus paracasei ATCC 334 in a cheese model system: A biochemical approach

Growth of Lactobacillus paracasei ATCC 334, in a cheese-ripening model system based upon a medium prepared from ripening Cheddar cheese extract (CCE) was evaluated. Lactobacillus paracasei ATCC 334 grows in CCE made from cheese ripened for 2 (2mCCE), 6 (6mCCE), and 8 (8mCCE) mo, to final cell densities of 5.9×10(8), 1.2×10(8), and 2.1×10(7)cfu/mL, respectively. Biochemical analysis and mass balance equations were used to determine substrate consumption patterns and products formed in 2mCCE. The products formed included formate, acetate, and D-lactate. These data allowed us to identify the pathways likely used and to initiate metabolic flux analysis. The production of volatiles during growth of Lb. paracasei ATCC 334 in 8mCCE was monitored to evaluate the metabolic pathways utilized by Lb. paracasei during the later stages of ripening Cheddar cheese. The 2 volatiles detected at high levels were ethanol and acetate. The remaining detected volatiles are present in significantly lower amounts and likely result from amino acid, pyruvate, and acetyl-coenzyme A metabolism. Carbon balance of galactose, lactose, citrate, and phosphoserine/phosphoserine-containing peptides in terms of D-lactate, acetate, and formate are in agreement with the amounts of substrates observed in 2mCCE; however, this was not the case for 6mCCE and 8mCCE, suggesting that additional energy sources are utilized during growth of Lb. paracasei ATCC 334 in these CCE. This study provides valuable information on the biochemistry and physiology of Lb. paracasei ATCC 334 in ripening cheese.

Diacetyl levels and volatile profiles of commercial starter distillates and selected dairy foods

Starter distillates (SDL) are used as ingredients in the formulation of many food products such as cottage cheese, margarine, vegetable oil spreads, processed cheese, and sour cream to increase the levels of naturally occurring buttery aroma associated with fermentation. This buttery aroma results, in part, from the presence of the vicinal dicarbonyl, diacetyl, which imparts a high level of buttery flavor notes and is a key component of SDL. Diacetyl (2,3-butanedione) is a volatile product of citrate metabolism produced by certain bacteria, including Lactococcus lactis ssp. diacetylactis and Leuconostoc citrovorum. In the United States, SDL are regarded as generally recognized as safe ingredients, whereby usage in food products is limited by good manufacturing practices. Recently, diacetyl has been implicated as a causative agent in certain lung ailments in plant workers; however, little is published about the volatile composition of SDL and the levels of diacetyl or other flavoring components in finished dairy products. The objective of this work was to characterize the volatile compounds of commercial SDL and to quantitate levels of diacetyl and other Flavor and Extract Manufacturers Association-designated high-priority flavoring components found in 18 SDL samples and 24 selected dairy products. Headspace volatiles were assessed using a solid-phase microextraction and analyzed by gas chromatography-mass spectrometry. In addition to diacetyl (ranging from 1.2 to 22,000 μg/g), 40 compounds including 8 organic acids, 4 alcohols, 3 aldehydes, 7 esters, 3 furans, 10 ketones, 2 lactones, 2 sulfur-containing compounds, and 1 terpene were detected in the SDL. A total of 22 food samples were found to contain diacetyl ranging from 4.5 to 2,700 μg/100g. Other volatile compounds, including acetaldehyde, acetic acid, acetoin, benzaldehyde, butyric acid, formic acid, furfural, 2,3-heptanedione, 2,3-pentanedione, and propanoic acid, were also identified and quantified in SDL or food samples, or both. The results obtained in this work summarize the volatile composition of commercial SDL and the approximate levels of diacetyl and other Flavor and Extract Manufacturers Association-designated high-priority flavoring components found in SDL and selected dairy foods.

Role of Cystathionine β-Lyase in Catabolism of Amino Acids to Sulfur Volatiles by Genetic Variants of Lactobacillus helveticus CNRZ 32

ABSTRACT Catabolism of sulfur-containing amino acids plays an important role in the development of cheese flavor. During ripening, cystathionine β-lyase (CBL) is believed to contribute to the formation of volatile sulfur compounds (VSCs) such as methanethiol and dimethyl disulfide. However, the role of CBL in the generation of VSCs from the catabolism of specific sulfur-containing amino acids is not well characterized. The objective of this study was to investigate the role of CBL in VSC formation by Lactobacillus helveticus CNRZ 32 using genetic variants of L. helveticus CNRZ 32 including the CBL-null mutant, complementation of the CBL-null mutant, and the CBL overexpression mutant. The formation of VSCs from methionine, cystathionine, and cysteine was determined in a model system using gas chromatography-mass spectrometry with solid-phase microextraction. With methionine as a substrate, CBL overexpression resulted in higher VSC production than that of wild-type L. helveticus CNRZ 32 or the CBL-null mutant. However, there were no differences in VSC production between the wild type and the CBL-null mutant. With cystathionine, methanethiol production was detected from the CBL overexpression variant and complementation of the CBL-null mutant, implying that CBL may be involved in the conversion of cystathionine to methanethiol. With cysteine, no differences in VSC formation were observed between the wild type and genetic variants, indicating that CBL does not contribute to the conversion of cysteine.

Reducing sodium levels in frankfurters using a natural flavor enhancer

Sodium chloride (NaCl; salt) contributes to important quality and food safety properties of processed meats. However, renewed interest exists in reducing sodium in the human diet. This study investigated quality and sensory impacts associated with partial replacement and/or reduction of normally added NaCl using a natural flavor enhancer (NFE) in frankfurters. Varying levels of NFE were used with NaCl and/or potassium chloride (KCl) to comprise treatments (TRT) which investigated flake salt replacement (Phase I) and sodium reduction (Phases II and III). Phase I sensory and quality results identified a 50% replacement of NaCl with NFE as the baseline for subsequent phases. Phase II indicated that the inclusion of NFE could allow for a 20% NaCl reduction without adverse effects on quality or sensory attributes. Phase III results demonstrated that it was feasible to reduce NaCl by 35% via the inclusion of KCl in NFE containing frankfurters without major quality or sensory changes.

Reducing sodium levels in frankfurters using naturally brewed soy sauce

Sodium chloride (NaCl; salt) serves important functions in processed meats, contributing to desirable quality and food safety characteristics; however, renewed interest exists in reducing sodium in the human diet despite sodium being a required component of the diet for physiological regulation. This study investigated consumer sensory and quality impacts from replacement of normally added NaCl (flake salt) with naturally brewed soy sauce (SS). Varying levels of SS were used with NaCl and/or potassium chloride (KCl) to comprise treatments (TRT) which investigated flake salt replacement (Phase I) and sodium reduction (Phases II and III). Phase I identified a 50% replacement of NaCl with SS as the baseline for subsequent phases. Phase II indicated that the inclusion of SS could allow for a 20% NaCl reduction without adverse effects on quality or sensory attributes. Phase III results demonstrated that it was feasible to reduce NaCl by 35% via the inclusion of KCl in SS containing frankfurters without major quality or sensory changes.