Gluconate metabolism and gas production by Paucilactobacillus wasatchensis WDC04

Paucilactobacillus wasatchensis, a nonstarter lactic acid bacteria, can cause late gas production and splits and cracks in aging cheese when it metabolizes 6-carbon substrates, particularly galactose, to a 5-carbon sugar, resulting in the release of CO₂. Previous studies have not explained late gas production in aging cheese when no galactose is present. Based on the genome sequence of Pa. wasatchensis WDC04, genes for potential metabolic pathways were mapped using knowledgebase predictive biology software. This metabolic modeling predicted Pa. wasatchensis WDC04 could metabolize gluconate. Gluconate contains 6 carbons, and Pa. wasatchensis WDC04 contains genes to convert it to 6-P-gluconate and then to ribulose-5-P by using 6-phosphogluconate dehydrogenase in a decarboxylating step, producing CO₂ during its metabolism. The goal of this study was to determine if sodium gluconate, often added to cheese to reduce calcium lactate crystal formation, could be metabolized by Pa. wasatchensis WDC04, resulting in gas production. Carbohydrate-restricted DeMan, Rogosa, and Sharpe broth was mixed with varying ratios of ribose, sodium gluconate, or d-galactose (total added substrate content of 1% wt/vol). Oxyrase (Oxyrase Inc.; 1.8% vol/vol) was also used to mimic the anaerobic environment of cheese aging in selected tubes. Tubes were inoculated with a 4-d culture of Pa. wasatchensis WDCO4, and results were recorded over 8 d. When inoculated into carbohydrate-restricted DeMan, Rogosa, and Sharpe broth containing only sodium gluconate as the added substrate, Pa. wasatchensis WDC04 grew, confirming gluconate utilization. Of the 10 ratios used, Pa. wasatchensis WDC04 produced gas in 6 scenarios, with the most gas production resulting from the ratio of 100% sodium gluconate with no added ribose or galactose. It was confirmed that obligately heterofermentative nonstarter lactobacilli such as Pa. wasatchensis WDC04 can utilize sodium gluconate to produce CO₂ gas. Addition of sodium gluconate to cheese thus becomes another risk factor for unwanted gas production and formation of slits and cracks.     

Galactose-positive adjunct cultures prevent gas formation by Paucilactobacillus wasatchensis WDC04 in a model gas production test

Gas production by obligatory heterofermentative lactic acid bacteria such as Paucilactobacillus wasatchensis is a sporadic problem in Cheddar cheese and results in undesired slits and cracks in the cheese. Growth of Pa. wasatchensis is not rapid, which makes investigations of gas production difficult to consistently execute. A primary objective of this study was to develop a model gas production test that could be used to investigate the effect of galactose and ribose utilization on gas production by Pa. wasatchensis and determine whether galactose-fermenting adjunct cultures could prevent gas formation. Paucilactobacillus wasatchensis WDC04 was inoculated at 10¹ to 10⁶ cfu/mL into carbohydrate-restricted MRS broth containing different ribose and galactose levels and incubated for up to 21 d at 23°C. Gas production in the broth was detected using a Durham tube inverted on a 6-cm-long capillary tube; cells were enumerated at 4, 8, and 12 d; and residual galactose was also measured. Gas production was sporadic except for when 10⁵ cfu/mL of Pa. wasatchensis WDC04 was inoculated into broth containing 0.3% ribose and 0.7% galactose. In those tubes, gas production was consistently observed after 8-d incubation, by which time galactose levels had decreased to 0.15%. Co-inoculation of Pa. wasatchensis WDC04 with as few as 10³ cfu/mL of a lactose-negative galactose-positive adjunct culture (Pediococcus acidilactici 23F, Lacticaseibacillus paracasei UW4, or Lactobacillus helveticus 7995) resulted in galactose depletion by d 4 and no observable gas production by d 12. With less galactose available to the slower-growing Pa. wasatchensis WDC04, its growth was limited to 10⁸ cfu/mL when any of the adjunct cultures was co-inoculated, compared with 10⁹ cfu/mL when grown on its own. We concluded that galactose-fermenting adjunct cultures have potential for preventing unwanted gas production in cheese by competition for resources and especially by removing the 6-carbon galactose before it can be utilized for energy by an obligatory heterofermentative lactobacilli such as Pa. wasatchensis and produce carbon dioxide.     

Hot topic: Geographical distribution and strain diversity of Lactobacillus wasatchensis isolated from cheese with unwanted gas formation

Lactobacillus wasatchensis, an obligate heterofermentative nonstarter lactic acid bacteria (NSLAB) implicated in causing gas defects in aged cheeses, was originally isolated from an aged Cheddar produced in Logan, Utah. To determine the geographical distribution of this organism, we isolated slow-growing NSLAB from cheeses collected in different regions of the United States, Australia, New Zealand, and Ireland. Seven of the cheeses showed significant gas defects and 12 did not. Nonstarter lactic acid bacteria were isolated from these cheeses on de Man, Rogosa, and Sharpe medium supplemented with ribose, a preferred substrate for Lb. wasatchensis. Identification was confirmed with 16S rRNA gene sequencing and the API50CH (bioMérieux, Marcy l'Etoile, France) carbohydrate panel. Isolates were also compared with one another by using repetitive element sequence-based PCR (rep-PCR). Lactobacillus wasatchensis was isolated only from cheeses demonstrating late-gas development and was found in samples from 6 of the 7 cheeses. This supports laboratory evidence that this organism is a causative agent of late gas production defects. The rep-PCR analysis produced distinct genetic fingerprints for isolates from each cheese, indicating that Lb. wasatchensis is found in several regions across the United States and is not a local phenomenon.     

Nonstarter lactic acid bacteria biofilms and calcium lactate crystals in Cheddar cheese

A sanitized cheese plant was swabbed for the presence of nonstarter lactic acid bacteria (NSLAB) biofilms. Swabs were analyzed to determine the sources and microorganisms responsible for contamination. In pilot plant experiments, cheese vats filled with standard cheese milk (lactose:protein = 1.47) and ultrafiltered cheese milk (lactose:protein = 1.23) were inoculated with Lactococcus lactis ssp. cremoris starter culture (8 log cfu/mL) with or without Lactobacillus curvatus or Pediococci acidilactici as adjunct cultures (2 log cfu/mL). Cheddar cheeses were aged at 7.2 or 10°C for 168 d. The raw milk silo, ultrafiltration unit, cheddaring belt, and cheese tower had NSLAB biofilms ranging from 2 to 4 log cfu/100 cm². The population of Lb. curvatus reached 8 log cfu/g, whereas P. acidilactici reached 7 log cfu/g of experimental Cheddar cheese in 14 d. Higher NSLAB counts were observed in the first 14 d of aging in cheese stored at 10°C compared with that stored at 7.2°C. However, microbial counts decreased more quickly in Cheddar cheeses aged at 10°C compared with 7.2°C after 28 d. In cheeses without specific adjunct cultures (Lb. curvatus or P. acidilactici), calcium lactate crystals were not observed within 168 d. However, crystals were observed after only 56 d in cheeses containing Lb. curvatus, which also had increased concentration of d(−)-lactic acid compared with control cheeses. Our research shows that low levels of contamination with certain NSLAB can result in calcium lactate crystals, regardless of lactose:protein ratio.     

Effect of Lactobacillus helveticus and Propionibacterium freudenrichii ssp. shermanii combinations on propensity for split defect in Swiss cheese

One of the least controlled defects in Swiss cheese is development of splits that appear during refrigerated storage after cheese is removed from the warm room. Such fissures, or cracks, in the body of the cheese can be as short as 1 cm, or long enough to span a 90-kg block. A 2 x 2 x 2 factorial experiment was used to determine the effect of different Lactobacillus helveticus/Propionibacterium freudenreichii ssp. shermanii starter culture combinations on the occurrence of split defect in Swiss cheese. Eights vats of cheese were made in summer and eight in winter. Each 90-kg block of cheese was cut into twenty-four 4-kg blocks and graded based on the presence of splits. Only small variations were found in the composition of cheeses made during the same season. There were no correlations between moisture, pH, fat, protein, calcium, lactose contents, D/L lactate ratio, or protein degradation that could be used to predict splits after 90 d of storage. However, cheese made in the summer had 2% higher moisture content and a greater prevalence of splits. There was a sixfold increase in amount of downgraded cheese between the best and worst culture combinations used during cheese manufacture. After 90-d storage, 14 to 90% of cheese had splits in the summer, and 1 to 6% in the winter. Split formation increased with time from 60 to 120 d of storage and extent of split formation was influenced by both the lactobacilli and propionibacteria cultures used.     

Influence of adjunct use and cheese microenvironment on nonstarter bacteria in reduced-fat cheddar-type cheese

This study investigated population dynamics of starter, adjunct, and nonstarter lactic acid bacteria (NSLAB) in reduced-fat Cheddar and Colby cheese made with or without a Lactobacillus casei adjunct. Duplicate vats of cheese were manufactured and ripened at 7 degrees C. Bacterial populations were monitored periodically by plate counts and by DNA fingerprinting of cheese isolates with the random amplified polymorphic DNA technique. Isolates that displayed a unique DNA fingerprint were identified to the species level by partial nucleotide sequence analysis of the 16S rRNA gene. Nonstarter biota in both cheese types changed over time, but populations in the Colby cheese showed a greater degree of species heterogeneity. The addition of the L. casei adjunct to cheese milk at 10(4) cfu/ml did not completely suppress "wild" NSLAB populations, but it did appear to reduce nonstarter species and strain diversity in Colby and young Cheddar cheese. Nonetheless, nonstarter populations in all 6-mo-old cheeses were dominated by wild L. casei. Interestingly, the dominant strains of L. casei in each 6-mo-old cheese appeared to be affected more by adjunct treatment and not cheese variety.     

Nonstarter lactic acid bacteria and aging temperature affect calcium lactate crystallization in cheddar cheese

The occurrence of unappetizing calcium lactate crystals in Cheddar cheese is a challenge and expense to manufacturers, and this research was designed to understand their origin. It was hypothesized that nonstarter lactic acid bacteria (NSLAB) affect calcium lactate crystallization (CLC) by producing D(-)-lactate. This study was designed to understand the effect of NSLAB growth and aging temperature on CLC. Cheeses were made from milk inoculated with Lactococcus lactis starter culture, with or without Lactobacillus curvatus or L. helveticus WSU19 adjunct cultures. Cheeses were aged at 4 or 13 degrees C for 28 d, then half of the cheeses from 4 and 13 degrees C were transferred to 13 and 4 degrees C, respectively, for the remainder of aging. The form of lactate in cheeses without adjunct culture or with L. helveticus WSU19 was predominantly L(+)-lactate (> 95%, wt/wt), and crystals were not observed within 70 d. While initial lactate in cheeses containingL. curvatus was only L(+)-lactate, the concentration of D(-)-lactate increased during aging. After 28 d, a racemic mixture of D/L-lactate was measured in cheeses containing L. curvatus; at the same time, CLC was observed. The earliest and most extensive CLC occurred on cheeses aged at 13 degrees C for 28 d then transferred to 4 degrees C. These results showed that production of D(-)-lactate by NSLAB, and aging temperature affect CLC in maturing Cheddar cheese.     

Effect of a bacteriocin‐producing strain of Lactobacillus paracasei on the nonstarter microflora of Cheddar cheese

In this study, the growth of chloramphenicol‐resistant bacteriocin‐sensitive indicator strain Lactobacillus casei DPC 2048^CM was evaluated in Cheddar cheese made with bacteriocin‐producing Lactobacillus paracasei DPC 4715. No suppression of growth of the indicator strain was observed in the cheese during ripening, and no bacteriocin production by L. paracasei DPC 4715 was detected by the well diffusion method in cheese and cheese extracts. The bacteriocin produced by L. paracasei DPC 4715 was sensitive to chymosin and cathepsin D, and it may have been hydrolysed by the rennet used for cheese manufacture or by indigenous milk proteases.     

Influence of calcium and phosphorus, lactose, and salt-to-moisture ratio on Cheddar cheese quality: proteolysis during ripening

Proteolysis in cheese is influenced by the state of proteins (protein-calcium-phosphate interactions), level of indigenous milk enzymes (plasmin), externally added milk-clotting enzymes (chymosin), and endogenous and exogenous enzymes from starter and non-starter lactic acid bacteria (NSLAB). The objective of this study was to determine how different levels of calcium (Ca) and phosphorus (P), residual lactose, and salt-to-moisture ratio (S/M) in cheese influence proteolysis during ripening. Eight cheeses with 2 levels of Ca and P (0.67 and 0.47% vs. 0.53 and 0.39%, respectively), 2 levels of lactose at pressing (2.4 vs. 0.78%), and 2 levels of S/M (6.4 vs. 4.8%) were manufactured. The cheeses were analyzed for changes in pH 4.6-soluble N, and starter and NSLAB counts during 48 wk of ripening. Cheeses at d 1 were also analyzed for residual chymosin, plasmin, and plasminogen activity. A significant increase in soluble N was observed during ripening for all the treatments. Cheeses with low Ca and P, low lactose, and low S/M treatments exhibited higher levels of proteolysis as compared to their corresponding high treatments. Differences in the rate of proteolysis for cheeses with different levels of Ca and P might be due to changes in protein conformation and differences in residual chymosin in the cheeses. Cheeses with low Ca and P were manufactured by lowering the pH at set and drain, which led to higher chymosin retention in cheeses with low Ca and P compared with high Ca and P. Differences in proteolysis between treatments with different levels of lactose were also partly attributed to residual chymosin activity. In all treatments, a major fraction of plasmin existed as plasminogen, indicating minimal contribution of plasmin to proteolysis in Cheddar cheeses. The number of starter bacteria, in all treatments, decreased significantly during ripening. However, the decrease was larger in the case of high S/M treatments compared with low S/M treatments. In contrast, the number of NSLAB increased during ripening, and low S/M cheeses had higher counts compared with high S/M cheeses. The differences in proteolysis due to S/M were partially attributed to changes in protein conformation or bacterial proteolytic activity.     

A factory-scale application of secondary adjunct cultures selected from lactic acid bacteria during Puzzone di Moena cheese ripening

The lactic acid populations of 2 seasonal Puzzone di Moena cheeses made from winter and summer raw cow's milk were characterized at different ripening times. Lactic acid bacteria (LAB) were isolated on selective media and subjected to genetic typing and identification. The species most frequently found during ripening were Lactobacillus paracasei ssp. paracasei, Lactobacillus plantarum, and Pediococcus pentosaceus. The different strains recognized by random amplification of polymorphic DNA-PCR were characterized for their acidifying and proteolytic activities to select nonstarter LAB to be used as secondary adjunct cultures (SAC). For each of the 3 above species, a strain showing weak acidification and high proteolytic capacity was selected. The 3 strains (Lb. paracasei ssp. paracasei P397, Lb. plantarum P399, and P. pentosaceus P41) constituted a mixed SAC used at 2 levels of concentration (10³ and 10⁴ cfu/mL) in experimental cheese making at dairy factory-scale. The analysis of volatile organic compounds as well as sensory analyses showed that the preferred level of SAC inoculation was 10³ cfu/mL.     

乳酸菌细菌素的高效表达方法研究

乳酸菌细菌素作为天然的生物防腐剂,因其高效、无毒等特点而备受关注,但目前只有乳酸链球菌素实现了工业化生产。乳酸菌细菌素产量低是限制其大规模生产的瓶颈之一。从高产菌株筛选、发酵条件调控、基因工程技术、诱导调控和胁迫刺激应答5个方面,综述了提高乳酸菌细菌素产量的方法,期望为提高乳酸菌细菌素产量的研究提供新思路。     

Effect of milk pasteurisation temperature on age-related changes in lactose metabolism,pH and the growth of non-starter lactic acid bacteria in half-fat Cheddar cheese

Half-fat Cheddar cheese (∼15%, w/w, fat) was manufactured on three occasions from milk pasteurised at 72, 77, 82 or 87 °C for 26 s, and analysed over a 270 day ripening period. Increasing milk pasteurisation temperature significantly increased the levels of moisture (from ∼45% at 72 °C to 50% at 87 °C), total lactate, and D(−)-lactate in cheese over the 270 day ripening period. Conversely, the cheese pH decreased significantly on increasing pasteurisation temperature. Increasing the pasteurisation temperature did not significantly affect the populations of starter or non-starter lactic acid bacteria during maturation. The use of higher pasteurisation temperatures would appear particularly amenable to exploitation as a means of producing high-moisture (e.g., 40–41%), short-ripened, mild-flavoured Cheddar or Cheddar-like cheeses.     

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 × 10⁸, 1.2 × 10⁸, and 2.1 × 10⁷ 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.     

葡萄糖酸钠对仔猪肠道内乳酸菌增殖及培养条件的研究

该研究采用平板计数方法,研究了葡萄糖酸钠添加对仔猪肠道内乳酸菌增殖的影响情况,研究结果表明,添加一定量的葡萄糖酸钠于仔猪饲料能促进肠道内的乳酸菌的生长,使其增殖.从仔猪肠道内分离纯化出一种乳酸杆菌,并研究了葡萄糖酸钠对该菌的体外促进作用,研究结果显示,低浓度的葡萄糖酸钠能促进乳酸菌的生长,高浓度的葡萄糖酸钠能抑制乳酸菌的生长.     

Real-time polymerase chain reaction for quantitative detection of histamine-producing bacteria: use in cheese production

Biogenic amines are toxic substances that appear in foods and beverages as a result of AA decarboxylation. The enzyme histidine decarboxylase catalyzes the decarboxylation of histidine to histamine, the biogenic amine most frequently involved in food poisoning. The aim of the present work was to develop a real-time quantitative PCR assay for the direct detection and quantification of histamine-producing strains in milk and cheese. A set of primers was designed, based on the histidine decarboxylase gene sequence of different gram-positive bacteria. The results show the proposed procedure to be a rapid (total processing time <2 h), specific and highly sensitive technique for detecting potential histamine-producing strains. Chromatographic methods (HPLC) verified the capacity of real-time quantitative PCR to correctly quantify histamine accumulation.     

发酵肉制品中乳酸菌的主要发酵特性

从发酵肉制品中分离、纯化乳酸菌132株,对它们的主要发酵特性如耐盐性、耐硝性、产黏液、产气等进行了测定,并比较了它们在不同培养基中的产酸情况。     

Angiotensin-converting enzyme inhibitory activity in Mexican Fresco cheese

The objective of this study was to evaluate if Mexican Fresco cheese manufactured with specific lactic acid bacteria (LAB) presented angiotensin I-converting enzyme inhibitory (ACEI) activity. Water-soluble extracts (3 kDa) obtained from Mexican Fresco cheese prepared with specific LAB (Lactococcus, Lactobacillus, Enterococcus, and mixtures: Lactococcus-Lactobacillus and Lactococcus-Enterococcus) were evaluated for ACEI activity. Specific peptide fractions with high ACEI were analyzed using reverse phase-HPLC coupled to mass spectrometry for determination of amino acid sequence. Cheese containing Enterococcus faecium or a Lactococcus lactis ssp. lactis-Enterococcus faecium mixture showed the largest number of fractions with ACEI activity and the lowest half-maximal inhibitory concentration (IC₅₀; <10 μg/mL). Various ACEI peptides derived from β-casein [(f(193-205), f(193-207), and f(193-209)] and α_S1-casein [f(1-15), f(1-22), f(14-23), and f(24-34)] were found. The Mexican Fresco cheese manufactured with specific LAB strains produced peptides with potential antihypertensive activity.     

Identification of aroma-active compounds in Cheddar cheese imparted by wood smoke

Cheddar cheese is the most popular cheese in the United States, and the demand for specialty categories of cheese, such as smoked cheese, are rising. The objective of this study was to characterize the flavor differences among Cheddar cheeses smoked with hickory, cherry, or apple woods, and to identify important aroma-active compounds contributing to these differences. First, the aroma-active compound profiles of hickory, cherry, and apple wood smokes were analyzed by solid-phase microextraction (SPME) gas chromatography-olfactometry (GCO) and gas chromatography-mass spectrometry (GC-MS). Subsequently, commercial Cheddar cheeses smoked with hickory, cherry, or apple woods, as well as an unsmoked control, were evaluated by a trained sensory panel and by SPME GCO and GC-MS to identify aroma-active compounds. Selected compounds were quantified with external standard curves. Seventy-eight aroma-active compounds were identified in wood smokes. Compounds included phenolics, carbonyls, and furans. The trained panel identified distinct sensory attributes and intensities among the 3 cheeses exposed to different wood smokes (P < 0.05). Hickory smoked cheeses had the highest intensities of flavors associated with characteristic “smokiness” including smoke aroma, overall smoke flavor intensity, and meaty, smoky flavor. Cherry wood smoked cheeses were distinguished by the presence of a fruity flavor. Apple wood smoked cheeses were characterized by the presence of a waxy, green flavor. Ninety-nine aroma-active compounds were identified in smoked cheeses. Phenol, guaiacol, 4-methylguaiacol, and syringol were identified as the most important compounds contributing to characteristic “smokiness.” Benzyl alcohol contributed to the fruity flavor in cherry wood smoked cheeses, and 2-methyl-2-butenal and 2-ethylfuran were responsible for the waxy, green flavor identified in apple wood smoked cheeses. These smoke flavor compounds, in addition to diacetyl and acetoin, were deemed important to the flavor of cheeses in this study. Results from this study identified volatile aroma-active compounds contributing to differences in sensory perception among Cheddar cheeses smoked with different wood sources.     

气相色谱法测定白酒中的乳酸

利用苄基溴对白酒中的乳酸进行酯化处理，以2-乙基正丁酸为内标，在HP-FFAP 19091F-413型毛细管柱上检测。实验结果表明，此法测定乳酸的相对标准偏差少于0.5%(n=7)，回收率为95%以上，线性范围为0-950mg/L，r=0.99975。该法简单、准确、重现性好。     

Molecular identification of naturally occurring bacteriocinogenic and bacteriocinogenic-like lactic acid bacteria in raw milk and soft cheese

Lactic acid bacteria (LAB) are currently used by food industries because of their ability to produce metabolites with antimicrobial activity against gram-positive pathogens and spoilage microorganisms. The objectives of this study were to identify naturally occurring bacteriocinogenic or bacteriocinogenic-like LAB in raw milk and soft cheese and to detect the presence of nisin-coding genes in cultures identified as Lactococcus lactis. Lactic acid bacteria cultures were isolated from 389 raw milk and soft cheese samples and were later characterized for the production of antimicrobial substances against Listeria monocytogenes. Of these, 58 (14.9%) LAB cultures were identified as antagonistic; the nature of this antagonistic activity was then characterized via enzymatic tests to confirm the proteinaceous nature of the antimicrobial substances. In addition, 20 of these antagonistic cultures were selected and submitted to genetic sequencing; they were identified as Lactobacillus plantarum (n = 2) and Lactococcus lactis ssp. lactis (n = 18). Nisin genes were identified by polymerase chain reaction in 7 of these cultures. The identified bacteriocinogenic and bacteriocinogenic-like cultures were highly variable concerning the production and activity of antimicrobial substances, even when they were genetically similar. The obtained results indicated the need for molecular and phenotypic methodologies to properly characterize bacteriocinogenic LAB, as well as the potential use of these cultures as tools to provide food safety.