Comparison of growth and survival of single strains of Lactococcus lactis and Lactococcus cremoris during Cheddar cheese manufacture

Traditionally, starter cultures for Cheddar cheese are combinations of Lactococcus lactis and Lactococcus cremoris. Our goal was to compare growth and survival of individual strains during cheesemaking, and after salting and pressing. Cultures used were 2 strains of L. lactis (SSM 7605, SSM 7436) and 2 strains of L. cremoris (SSM 7136, SSM 7661). A standardized Cheddar cheese make procedure was used that included a 38°C cook temperature and salting levels of 2.0, 2.4, 2.8, 3.2, and 3.6% from which were selected cheeses with salt-in-moisture levels of 3.5, 4.5, and 5.5%. Vats of cheese were made using each strain on its own as biological duplicates on different days. Starter culture numbers were enumerated by plate counting during cheesemaking and after 6 d storage at 6°C. Flow cytometry with fluorescent staining by SYBR Green and propidium iodide was used to determine the number of live and dead cells in cheese at the different salt levels. Differences in cheese make times were strain dependent rather than species dependent. Even with correction for average culture chain length, cheeses made using L. lactis strains contained ～4 times (～0.6 log) more bacterial cells than those made using L. cremoris strains. Growth of the strains used in this study was not influenced by the amount of salt added to the curd. The higher pH of cheeses with higher salting levels was attributed to those cheeses having a lower moisture content. Based on flow cytometry, ～5% of the total starter culture cells in the cheese were dead after 6 d of storage. Another 3 to 19% of the cells were designated as being live, but semipermeable, with L. cremoris strains having the higher number of semipermeable cells.     

Interactions between EPS-producing Streptococcus thermophilus strains in mixed yoghurt cultures

Mixed cultures of different EPS-producing Streptococcus thermophilus strains in combination with a Lactobacillus delbrueckii subsp. bulgaricus strain with negligible EPS-production were used for yoghurt production. The yoghurt texture was characterised with respect to sensory, rheological and microstructural properties and the EPS-concentrations were determined. The cultures resulted in yoghurts with highly different texture properties, and positive interactions between certain Streptococcus thermophilus strains were observed. The underlying properties of yoghurt texture are multidimensional, but a number of microstructural characteristics were apparent in the yoghurts with the highest mouth thickness, creaminess and viscosity. A strong protein network, not too dense and with medium size pores containing EPS, seems associated with these properties. The presence of capsular polysaccharides (CPS) also appeared to be beneficial as did a combination of EPS types, which were distributed differently in the protein network (in serum pores, respectively in association with protein). Obviously, a certain concentration of EPS must be present to provide for these effects on yoghurt texture, but other factors than concentration per se seem more important.     

Impact of ropy and capsular exopolysaccharide-producing strains of Lactococcus lactis subsp. cremoris on reduced-fat Cheddar cheese production and whey composition

Cheddar cheese mixed starter cultures containing exopolysaccharide (EPS)-producing strains of Lactococcus lactis subsp. cremoris (Lac. cremoris) were characterized and used for the production of reduced-fat Cheddar cheese (15% fat). The effects of ropy and capsular strains and their combination on cheese production and physical characteristics as well as composition of the resultant whey samples were investigated and compared with the impact of adding 0.2% (w/v) of lecithin, as a thickening agent, to cheese milk. Control cheese was made using EPS-non-producing Lac. cremoris. Cheeses made with capsular or ropy strains or their combination retained 3.6–4.8% more moisture and resulted in 0.29–1.19 kg/100 kg higher yield than control cheese. Lecithin also increased the moisture retention and cheese yield by 1.4% and 0.37%, respectively, over the control cheese. Lecithin addition also substantially increased viscosity, total solid content and concentrating time by ultra-filtration (UF) of the whey produced. Compared with lecithin addition, the application of EPS-producing strains increased the viscosity of the resultant whey slightly, while decreasing whey total solids, and prolonging the time required to concentrate whey samples by UF. The amount of EPS expelled in whey ranged from 31 to 53 mg L⁻¹. Retention of EPS-producing strains in cheese curd was remarkably higher than that of non-producing strains. These results indicate the capacity of EPS-producing Lac. cremoris for enhanced moisture retention in reduced-fat Cheddar cheese; these strains would be a promising alternative to commercial stabilizers.     

Formation of methional by Lactococcus lactis IFPL730 under cheese model conditions

The present work describes the capacity of Lactococcus lactis to produce methional and other sulphur compounds derived from methionine (Met) in a cheese model system. Cheese slurries were prepared from pasteurized ewes' skimmed milk, chemically acidified with glucono-'-lactone and homogenized aseptically adding Met, !-ketoglutarate, pyridoxal 5'-phosphate, thiamine pyrophosphate and NaCl. Slurries were incubated at 12 °C for 14 days with cellular suspensions of L. lactis IFPL359, L. lactis IFPL730 and L. lactis NCDO763 in different combinations. Slurries added with resting cells and the intracellular fraction from L. lactis IFPL730 showed the highest production of methional at the outset of incubation, which decreased during incubation along with a concomitant increase in 3-methylthiopropanol. The sensorial analysis of slurries indicated a characteristic methional aroma (cooked potato-like) in samples containing 4-methylthio-2-ketobutyrate and the intracellular fraction from L. lactis IFPL730. As incubation proceeded, the intensity of methional aroma decreased but samples were judged by the panel tasters as developing a cheese-like flavour.     

Effects of high pressure treatment on glycolytic enzymes of Lactococcus lactis subsp. lactis,Streptococcus thermophilus and Lactobacillus acidophilus

High pressure processing (HPP) reduces the glycolytic activity of lactic acid bacteria (LAB) and provides a means to control further production of acidic metabolites in fermented dairy products during storage. However, there is limited information on the effects of HPP on specific enzymes of dairy starter bacteria responsible for the metabolism of lactose. The aim of this study was to determine pressure-induced inactivation of glycolytic enzymes in Lactococcus lactis subsp. lactis C10, Streptococcus thermophilus TS1 and Lactobacillus acidophilus 2400. Cultures were grown for 16 h in M17 or MRS broth containing 5% (w/v) lactose at pH 6.5 (maintained by addition of 10 M NaOH). The cells were harvested by centrifugation, washed and resuspended in 100 mM phosphate buffer (pH 6.5) and pressure-treated at 300 and 600 MPa (≤ 22 °C, 5 min). The ability of pressure-treated resting cells of Lactococcus, incubated with 5% (w/v) lactose at 30 °C, to ferment lactose was evaluated by determining titratable acidity (TA) during incubation. The activities of phospho-β-galactosidase (P-β-gal), β-galactosidase (β-gal) and lactate dehydrogenase (LDH) were determined in cell-free extracts of untreated and pressure-treated cells. Resting cells of Lactococcus treated at 600 MPa had a substantially lower rate of acidification than the controls and those treated at 300 MPa. Both P-β-gal and β-gal were significantly inactivated (p < 0.01) in the starter cultures treated at 300 or 600 MPa. The LDH in Lactococcus and Lactobacillus was highly resistant to pressure treatment at 300 MPa. In contrast, the LDH in Streptococcus was almost completely inactivated at ≥ 300 MPa.Industrial relevanceContinuing production of acidic metabolites in fermented dairy products during storage can be a technological challenge that adversely affects product quality. The current study demonstrates that high pressure processing (HPP) offers the potential of controlling this problem by inactivation of glycolytic enzymes in various mesophilic and thermophilic starter cultures. The findings of this research will assist in establishing optimised operating parameters for HPP treatment of cultured products to extend shelf-life, by reducing acid production during storage.     

Thermal inactivation of the heat-resistant Lactococcus lactis bacteriophage P680 in modern cheese processing

The thermal inactivation of the highly thermo-resistant test phage (P680), was investigated in whey, whey protein concentrate (WPC) and whey cream. After a heat treatment at 90 °C for 15 min, only a 6-log reduction was obtained and the phage was still detectable in each medium. Kinetic parameters for the inactivation of the phage were calculated for temperatures ranging from 70 to 90 °C using a non-linear model. With the help of the parameters obtained, the lines of equal effects showing a 9-log inactivation of the phage were calculated. High temperature short time pasteurization was not sufficient for 9-log inactivation of phage P680 in skim milk, whey, WPC or whey cream. Temperature and time combinations ranging from 100 °C for 20 min to 140 °C for 2 s are necessary for a 9-log inactivation of P680.     

Influence of proteolytic Lactococcus lactis subsp. cremoris on ripening of reduced-fat Cheddar cheese made with camel chymosin

This study investigated proteolysis in reduced-fat Cheddar cheese produced with camel chymosin and Lactococcus lactis subsp. cremoris with the ability to cleave the N-terminus of α_S1-casein. The aim was to match the activity of bovine chymosin, which leads to softer cheese structure than camel chymosin. Cheeses were analysed for gross composition, casein and peptide breakdown, release of free amino acids, structure parameters and sensory characteristics. Selected Lc. lactis subsp. cremoris increased the amount of peptides and, to a limited extent, the total amount of free amino acids in the cheeses. One group of experimental cheeses was found to have a significantly firmer structure, higher stress at fracture and modulus of deformability than the reference cheeses. The addition of the selected proteolytic dairy strains of Lc. lactis subsp. cremoris to the cheeses did not result in extended breakdown of α_S1-casein or a softer cheese structure.     

Technological characterization of bacteriocin producing Lactococcus lactis strains employed to control Listeria monocytogenes in cottage cheese

In recent years, there has been a particular focus on the application of antimicrobial compounds produced by lactic acid bacteria (LAB) as natural preservatives to control the growth of spoilage and pathogenic bacteria in food. Bacteriocins are antimicrobial peptides which can be added to foods in concentrated forms as food preservatives, e.g. additives, or they can be produced in situ by starters or protective cultures. In this study, twenty Lactococcus lactis bacteriocin producers previously isolated from Italian fermented foods were subjected to a variety of physical and biochemical tests in order to identify those with the greatest potential as starter cultures in cheese production. Of these, four strains isolated from cheese (one nisin Z producer, one nisin A producer and two lacticin 481 producers) which fulfilled the desired technological criteria were assessed for their ability to control Listeria monocytogenes. The subsequent application of these bacteriocinogenic strains as starter cultures in Cottage cheese established that the nisin A producing Lact. lactis 40FEL3, and to a lesser extent the lacticin 481 producers 32FL1 and 32FL3, successfully controlled the growth of the pathogen. This is the first study to directly compare the ability of nisin A, nisin Z and lacticin 481 producing strains to control listerial growth during the manufacture and storage of Cottage cheese.     

Characterization of Streptococcus thermophilus strains that undergo lysis under unfavourable environmental conditions

The autolysis of starter lactic acid bacteria appears as a promising way to enhance the flavour of fermented dairy products. The present work was aimed at investigating the autolysis phenomenon in Streptococcus thermophilus, a thermophilic lactic acid bacteria involved in the starters used for the production of yoghurts, Italian and Swiss-type cheeses. Out of 146 strains screened for their aptitude to spontaneously lyse at the end of growth in M17 medium containing lactose in limited concentration, six strains, among which is the type strain CNRZ 1358, were found to be highly autolytic. These autolytic strains are characterized by a typical bell-shaped growth curve. Lysis of the type strain, which was studied as the model, was triggered under unfavourable environmental conditions, such as lactose depletion and NaCl or organic solvents addition. The lysogenic character of this strain was evidenced. Taken together, our results indicate that the autolytic phenotype in S. thermophilus is linked to the lysogenic character but does not result from the massive prophage induction under stressing conditions.     

Citrate metabolism in Lactococcus lactis subsp. lactis var. diacetylactis strains

The formation of diacetyl, acetoin, 2,3-butylene glycol, acetaldehyde, ethanol and lactic acid during 24 h of cultivation in milk with 0.19 and 0.5 % of citrate has been studied. Depending on the strain, bacteria produced 1.5 - 1.9 mg of diacetyl, 212 - 311 mg of acetoin and 137 - 156 mg of butylene glycol in 1 1 milk. An increase of the citrate concentration in milk to 0.5 % resulted in an increase in the production of diacetyl from 58 to 74 % and of acetoin by 2.8 - 3.7 times. The strains of distinct activity of acetoin reductase produced in these conditions 2.3 - 2.7 times as much as 2,3-butylene glycol. The recovery of citrate in the from of C₄-compounds ranged from 76 to 98 %, yet barely 0.18 - 0.44 % in the from of diacetyl. Increased concentration of citrate in milk stimulated the production of diacetyl and acetaldehyde to the similar extent, thereby it did not result in the deterioration of organoleptic qualities of starters and milk products. Within the doses used citrate did not significantly affect growth and acidifying activity of the bacteria.     

Phenotypic and genetic characterization of a selected set of Lactococcus lactis strains isolated from a starter-free farmhouse cheese

Eleven strains of lactococci isolated from a farmhouse starter-free cheese manufactured from raw cow's milk were analysed in detail for some technologically-related properties. Large phenotypic differences were encountered between the isolates, some of which could be of practical relevance. Several strains produced lactic acid at a rate and at a final concentration suitable for large-scale cheesemaking. The enzymatic capabilities assayed with the API ZYM system showed that all strains possess similar profiles with weak proteinase and moderate leucine-arylamidase and esterase-lipase activities. Interestingly, two related strains presented a strong β -galactosidase activity. Plasmid and chromosomal analyses indicated a high degree of diversity among wild strains and showed low homology with some well-known Lactococcus lactis strains. Under highly stringent conditions, only one plasmid from a single strain gave a clear positive hybridization signal with lactococcal-derived probes for the β -phospho- galactosidase and the proteinase genes. In general, wild strains produced more odorous compounds and in higher amounts than the reference strains.     

切达干酪发酵菌种的特性测定及发酵干酪的评价

对实验室研究较为成熟的2株乳酸乳球菌进行发酵产酸、产黏测定,同时对其蛋白水解能力进行评价;并对单菌株、不同比例组合菌株制备的切达干酪进行质构特性评价、感官分析和风味物质测定。测定结果表明:乳酸乳球菌乳酸亚种KLDS4.0424是主要的产酸菌种;与商业发酵剂相比,实验室菌株的蛋白水解能力和产黏能力相对较弱;乳酸乳球菌乳酸亚种KLDS4.0424与乳酸乳球菌乳脂亚种KLDS4.0326按1∶1接种制作的干酪具有良好的成熟度、质地和风味,具备开发成干酪发酵剂的潜力。      

Influence of capsular and ropy exopolysaccharide-producing Streptococcus thermophilus on Mozzarella cheese and cheese whey

We investigated the effect of capsular and ropy exopolysaccharide-producing Streptococcus thermophilus starter bacteria on Mozzarella cheese functionality and whey viscosity. Mozzarella cheeses were manufactured with Lactobacillus helveticus LH100 paired with one of four S. thermophilus strains: MR-1C, a bacterium that produces a capsular exopolysaccharide; MTC360, a strain that secretes a ropy exopolysaccharide; TAO61, a nonexopolysaccharide-producing commercial cheese starter; and DM10, a nonencapsulated, exopolysaccharide-negative mutant of strain MR-1C. As expected, cheese moisture levels were significantly higher in Mozzarella cheeses made with exopolysaccharide-positive versus exopolysaccharide-negative streptococci, and melt properties were better in the higher moisture cheeses. Whey viscosity measurements showed that unconcentrated and ultrafiltered, fivefold concentrated whey from cheeses made with S. thermophilus MTC360 were significantly more viscous than whey from cheeses made with MR-1C, TAO61, or DM10. No significant differences were noted between the viscosity of unconcentrated or concentrated whey from cheeses made with S. thermophilus MR-1C versus the industrial cheese starter TAO61. These data indicate that encapsulated, but not ropy, exopolysaccharide-producing S. thermophilus strains can be utilized to increase the moisture level of cheese and to improve the melt properties of Mozzarella cheese without adversely affecting whey viscosity.     

玉米胚芽蛋白Cheddar干酪生产工艺的优化

以玉米胚茅蛋白为添加辅料,研究了玉米胚芽蛋白添加量、Ca^2＋浓度、切割大小、盐浓度四个工艺参数对玉米胚芽Cheddar干酪品质的影响,利用响应面分析法确定了最佳工艺参数。结果表明,胚芽蛋白添加量为4．28％,Ca^2＋浓度为0．04％,切割大小为9．05mm3,盐浓度为1．62％时,生产的玉米胚芽Cheddar干酪具有干酪特有的滋味和气味,软硬适度、香味醇厚、营养丰富。     

Effect of galactose metabolising and non-metabolising strains of Streptococcus thermophilus as a starter culture adjunct on the properties of Cheddar cheese made with low or high pH at whey drainage

Cheddar cheese was made using control culture (Lactococcus lactis subsp. lactis), or with control culture plus a galactose-metabolising (Gal⁺) or galactose-non-metabolising (Gal⁻) Streptococcus thermophilus adjunct; for each culture type, the pH at whey drainage was either low (pH 6.15) or high (pH 6.45). S. thermophilus affected the levels of residual lactose and galactose, and the volatile compound profile and sensory properties of the mature cheese (270 d) to an extent dependent on the drain pH and phenotype (Gal⁺ or Gal⁻). For all culture systems, reducing drain pH resulted in lower levels of moisture and lactic acid, a higher concentration of free amino acids, and higher firmness. The results indicate that S. thermophilus may be used to diversify the sensory properties of Cheddar cheese, for example from a fruity buttery odour and creamy flavour to a more acid taste, rancid odour, and a sweaty cheese flavour at high drain pH.     

Interactions between milk proteins and exopolysaccharides produced by Lactococcus lactis observed by scanning electron microscopy

The interactions between exopolysaccharides produced by Lactococcus lactis ssp. cremoris JFR1 and dairy proteins (caseins and whey proteins) in fermented media (milk permeate and buttermilk) were observed using scanning electron microscopy. An immobilization technique by crosslinking was employed to bind the protein to the observation surface, so that a washing step could be performed to remove noninteracting material. The use of this novel technique allowed us, for the first time, to confirm that the exopolysaccharide molecules interact with dairy proteins. Exopolysaccharides appear as filament strands attached to the protein aggregates and to the bacterial cells. This new sample preparation technique proved to be very valuable for observing molecular interactions in fermented media.     

Efficacy of four conjugal lactococcal phage resistance plasmids against phage in commercial Lactococcus lactis subsp. cremoris cheese starter strains

The efficacy of four lactococcal phage resistance plasmids (pNP40, pMU1311, pDI60 and pKP100) against phage was assessed after their conjugal transfer to four commercial Lactococcus lactis subsp. cremoris cheese starter strains and to the plasmid-free strain L. lactis subsp. cremoris MG1363. In MG1363, only pNP40 conferred resistance to prolate phages c2 and 643. Highest levels of resistance to small isometric phages in MG1363 occurred when pNP40 was stacked together with pMU1311 or pDI60. In the four starter strains, the plasmids conferred varying levels of resistance to small isometric phages. Growth and acidification rates in milk of most transconjugants derived from the starter strains decreased, but this was not always due to loss of plasmid-encoded cell wall proteinase (lactocepin) activity. Only one transconjugant grew during repeated subculture in milk with addition of factory wheys containing phages. This and the presence of bacteriocins encoded on pMU1311 and pDI60 limited application of the plasmids to protect L. lactis subsp. cremoris starters against phages in industry. However, some of the plasmids could be useful in extending the industry life of starters where fast acid production is not required or where bacteriocin production is acceptable.     

Effect of wild strains of Lactococcus lactis on the volatile profile and the sensory characteristics of ewes' raw milk cheese

The production of volatile compounds by wild strains of Lactococcus lactis used as starter cultures and their effect on the sensory characteristics of ewes' raw milk cheese were investigated. Sixteen vats of cheese were manufactured and ripened for 120 d in two experiments, each of them duplicated. In the first experiment, milk was inoculated with different ratios of four wild Lactococcus lactis strains, two producing and two not producing branched-chain volatile compounds, and in the second experiment with different ratios of a commercial starter culture and the two strains producing branched-chain volatile compounds. Cheese pH, proteolysis, and aminopeptidase activity increased when the strains producing branched-chain volatile compounds were inoculated at a higher rate. Fifty volatile compounds were identified in cheeses using a purge and trap system coupled to a gas chromatography-mass spectrometry apparatus. The relative abundances of 30 volatile compounds (8 alcohols, 5 aldehydes, 3 ketones, 12 esters, 1 sulfur compound, and 1 benzenic compound) were influenced by starter culture composition. 2-Methylpropanol, 3-methylbutanol, isobutyl acetate, isoamyl acetate, ethyl butyrate, isobutyl butyrate, and isoamyl butyrate were always more abundant in the cheeses made with a higher level of L. lactis strains producing branched-chain volatile compounds. Flavor intensity was enhanced by a high level of L. lactis strains producing branched-chain volatile compounds in the first experiment, in which four wild L. lactis strains were used as starter culture, but not in the second experiment, in which a combination of two wild L. lactis strains and the commercial starter culture were used. Flavor quality, as judged by trained panelists, was impaired in both experiments by a high level of L. lactis strains producing branched-chain volatile compounds.