Influence of Homofermentative Lactobacilli on the Microflora and Soluble Nitrogen Components in Cheddar Cheese

Cheddar cheese was manufactured by inoculation with and without cultures of homofermentative Lactobacillus strains (L. casei-subsp-casei, L. casei-subsp-pseudoptantarum, L. plantarum). Growth rate of microflora, the incidence of heterofermentative lactobacilli and the rate of proteolysis were then studied during aging. The total number of psychotrophs, mesophiles and lactic streptococci reached a maximum at 5 months but maximum numbers attained were dependent upon the curing temperature (7°C or 15°C). The acceleration of cheese ripening by Lactobacillus cultures was accompanied by a greater degree of protein hydrolysis which was detectable after 8 months aging as soluble nitrogen in TCA extracts (360 mg/100g at 15°C and 240 mg at 7°C, versus 170 mg and 75 mg, respectively, for the controls).     

Use of high pressure treatment to attenuate starter bacteria for use as adjuncts for Cheddar cheese manufacture

Attenuated starter bacteria cannot produce acid during cheese manufacture, but contain enzymes that contribute to cheese ripening. The aim of this study was to investigate attenuation of starter bacteria using high pressure treatment, for use in combination with a primary starter for Cheddar cheese manufacture, and to determine the effect of such adjunct cultures on secondary proteolysis during ripening. Lactococcus lactis ssp. cremoris HP and L. lactis ssp. cremoris 303 were attenuated by pressure treatment at 200 MPa for 20 min at 20 °C. Cheddar cheese was manufactured using untreated cultures of both these starter strains, either alone or in combination with their high pressure-treated equivalents. High pressure-treated starters did not produce acid during cheese manufacture and starter counts in cheeses manufactured using high pressure-treated starter did not differ from those of the controls. Higher levels of cell lysis were apparent in cheese manufactured using high pressure-treated strains than in the controls after 26 d of ripening. Small differences were observed in the peptide profiles of cheeses, analysed by reversed-phase HPLC; cheeses manufactured using high pressure-treated starters also had slightly higher levels of amino acids than the relevant controls. Overall, addition of high pressure-treated starter bacteria as a secondary starter culture accelerated secondary proteolysis in Cheddar cheese.

Industrial relevance

Attenuated starters provide extra pool of enzymes, which can influence cheese ripening, without affecting the cheese making schedule. This paper presents an alternative method for attenuation of starter bacteria using high pressure treatment and their subsequent use to accelerate secondary proteolysis in Cheddar cheese during ripening.     

Isolation and characterisation of exopolysaccharide-producing Weissella and Lactobacillus and their application as adjunct cultures in Cheddar cheese

This study characterised exopolysaccharide-producing lactic acid bacteria and examined their potential for use in Cheddar cheese manufacture. Two strains were chosen for incorporation as adjunct cultures in Cheddar cheese manufacture: namely, the homopolysaccharide-producers Weissella cibaria MG1 and Lactobacillus reuteri cc2. These strains both produce dextrans with molecular masses ranging from 10⁵ to 10⁷ Da. Both strains were used in the production of miniature Cheddar cheeses that employed a conventional commercial cheese starter culture Lactococcus lactis R604. A cheese was also included that used purified dextran as an ingredient. The W. cibaria strain survived in cheese with levels increasing by 1.5 log cycles over the ripening period. All experimental cheeses (adjunct or exopolysaccharide ingredient) had higher moisture levels compared with the control cheese made using starter alone. Inclusion of the adjunct strains had no detectable negative effects on cheeses in terms of proteolysis.     

Evolution of Free Amino Acids during the Ripening of Cheddar Cheese Containing Added Lactobacilli Strains

Cheddar cheese was produced with different lactobacilli strains added to accelerate ripening. The concentration of proteolytic products was determined as free amino acids in the water-soluble fraction at two, four, seven and nine months of aging and at two different maturation temperatures (6°C, 15°C). All amino acids increased during ripening and were higher in the Lactobacillus- added cheeses than in the control cheese, and higher in cheeses ripened at 15°C than at 6°C. Glutamic acid, leucine, phenylalanine, valine and lysine were generally in higher proportion in all cheeses. The cheeses with added L. casei-casei L2A were classified as having a “strong Cheddar cheese” flavor after only seven months of ripening at 6°C.     

Effect of Free or Encapsulated Recombinant Aminopeptidase of Lactobacillus rhamnosus S93 on Acceleration of Cheddar Cheese Ripening

The use of recombinant aminopeptidase (PepN) from Lactobacillus rhamnosus S93 in free or encapsulated form was investigated to shorten the duration of Cheddar cheese ripening. Proteolysis was determined by measuring the soluble nitrogen as phosphotungstic acid (PTA-N) derivatives and free amino acids (FAA) over a 6-month period. The experimental cheeses received higher scores for sensory properties than the control cheese. The amounts of PTA-N and total FAA in the cheese with the encapsulated enzyme after 2 months of ripening were close to those of the control cheese after 6 months, suggesting the acceleration in proteolysis by about 4 months.     

Influence of Homofermentative Lactobacilli on Physicochemical and Sensory Properties of Cheddar Cheese

Cheddar cheeses were produced under pilot plant conditions using a commercial Streptococcus culture amended with one of 10 homofermentative Lactobacillus strains. During the ripening period, pH, acidity, salt, moisture, fat, texture, fissure formation, gas development and sensory status were evaluated. Lactobacillus treated cheese did not differ much from the control in pH and acidity but acidity increased substantially after draining and cheddaring. Lactobacillus numbers increased at all stages as compared with the uninoculated control. High quality Cheddar cheese was produced by L. casei-subsp-casei (119-10/62) and L. casei-subsp-pseudoplantarum (137-10/62) from 7 to 12 vats aged for 2 months at 15°C and for a further 10 months at 7°C or 15°C. Fissure formation was observed in cheese made with L. casei-subsp-rhamnosus, one of the four cultures of L. casei-subsp-casei (LH13) and two of the three strains of L. casei-subsp-pseudoplantarum (83-4-12/62 and L3E). Certain Lactobacillus strains produced cheese with slight flavor defects. Other strains, in particular L. casei-subsp-rhamnosus, contributed to high acidity (72 - 0.89° domic) and low pH (5.2) at salting.     

Application of exopolysaccharide-producing cultures in reduced-fat Cheddar cheese: composition and proteolysis

Proteolysis during ripening of reduced fat Cheddar cheeses made with different exopolysaccharide (EPS)-producing and nonproducing cultures was studied. A ropy strain of Lactococcus lactis ssp. cremoris (JFR1) and capsule-forming nonropy and moderately ropy strains of Streptococcus thermophilus were used in making reduced-fat Cheddar cheese. Commercial Cheddar starter was used in making full-fat cheese. Results showed that the actual yield of cheese made with JFR1 was higher than that of all other reduced-fat cheeses. Cheese made with JFR1 contained higher moisture, moisture in the nonfat substance, and residual coagulant activity than all other reduced-fat cheeses. Proteolysis, as determined by PAGE and the level of water-soluble nitrogen, was also higher in cheese made with JFR1 than in all other cheeses. The HPLC analysis showed a significant increase in hydrophobic peptides (causing bitterness) during storage of cheese made with JFR1. Cheese made with the capsule-forming nonropy adjunct of S. thermophilus, which contained lower moisture and moisture in the nonfat substance levels and lower chymosin activity than did cheese made with JFR1, accumulated less hydrophobic peptides. In conclusion, some EPS-producing cultures produced reduced-fat Cheddar cheese with moisture in the nonfat substance similar to that in its full-fat counterpart without the need for modifying the standard cheese-making protocol. Such cultures might accumulate hydrophobic (bitter) peptides if they do not contain the system able to hydrolyze them. For making high quality reduced-fat Cheddar cheese, EPS-producing cultures should be used in conjunction with debittering strains.     

Impact of Using Exopolysaccharides (EPS)-Producing Strain on Qualities of Half-Fat Cheddar Cheese

Yield, textural, proteolysis, melting, and sensory properties of exopolysaccharide-producing Lactobacillus paracasei on properties of half-fat (about 16 g fat/100 g cheese) Cheddar cheese during ripening at 8℃ for up to six months were investigated. The results revealed that B-3 cheese, made with 2.0% (v/v) high yield exopolysaccharide-producing L. paracasei in combination with 0.011% (w/w) commercial Cheddar culture (B-3 cheese), had a 10.15, 7.71, and 10.04% separately increase in moisture content and had a 7.70, 5.05, and 6.76% separately increase in yield compared with B-2, B-4, and B-5 cheese, texture and melting characteristics were significantly improved (P < 0.05), sensory score surpassed B-4 and B-5 cheese and was similar to the full-fat one. Any differences of B-3 cheese detected among half-fat Cheddar cheeses were attributed to the presence of high yield exopolysaccharide-producing L. paracasei.     

Impact of liposome-encapsulated enzyme cocktails on cheddar cheese ripening

Cheddar cheese proteolysis and lipolysis were accelerated using liposome-encapsulated enzymatic cocktails. Flavourzyme, neutral bacterial protease, acid fungal protease and lipase (Palatase M) were individually entrapped in liposomes and added to cheese milk prior to renneting. Flavourzyme was tested alone at three concentrations (Z1, Z2 and Z3 cheeses). Enzyme cocktails consisted of lipase and bacterial protease (BP cheeses), lipase and fungal protease (FP cheeses) or lipase and Flavourzyme (ZP cheeses). The resulting cheeses were chemically, rheologically and organoleptically evaluated during 3 months of ripening at 8 °C. Levels of free fatty acids and appearance of bitter and astringent peptides were measured. Certain enzyme treatments (BP and ZP) resulted in cheeses with more mature texture and higher flavor intensity in a shorter time compared with control cheeses. No bitter defect was detected except in 90-day-old FP cheese. A full aged Cheddar flavor was developed in Z3 and ZP cheeses, while treatment BP led to strong typical Cheddar flavor by the second month and did not exhibit any off-flavor when ripening was extended for a further month.     

Impact of nisin producing culture and liposome-encapsulated nisin on ripening of Lactobacillus added-Cheddar cheese

This study aimed to evaluate the effects of incorporating liposome-encapsulated nisin Z, nisin Z producing Lactococcus lactis ssp. lactis biovar. diacetylactis UL719, or Lactobacillus casei-casei L2A adjunct culture into cheese milk on textural, physicochemical and sensory attributes during ripening of Cheddar cheese. For this purpose, cheeses were made using a selected nisin tolerant cheese starter culture. Proteolysis, free fatty acid production, rheological parameters and hydrophilic/hydrophobic peptides evolution were monitored over 6 mo ripening. Sensory quality of cheeses was evaluated after 6 mo. Incorporating the nisin-producing strain into cheese starter culture increased proteolysis and lipolysis but did not significantly affect cheese rheology. Liposome-encapsulated nisin did not appear to affect cheese proteolysis, rheology and sensory characteristics. The nisinogenic strain increased the formation of both hydrophilic and hydrophobic peptides present in the cheese water extract. Sensory assessment indicated that acidic and bitter tastes were enhanced in the nisinogenic strain-containing cheese compared to control cheese. Incorporating Lb. casei and the nisinogenic culture into cheese produced a debittering effect and improved cheese flavor quality. Cheeses with added Lb. casei and liposome-encapsulated nisin Z exhibited the highest flavor intensity and were ranked first for sensory characteristics.     

Monitoring Proteolysis During Cheddar Cheese Ripening Using Two-Dimensional Gel Electrophoresis

A 2-D gel electrophoretic method, consisting of isoelectric focusing and alkaline urea-PAGE was used to monitor proteolysis during ripening (180d, 5°C and 8°C) of full- and reduced-fat Cheddar cheese. The method enabled quantifying changes in levels of peptides in cheese with good spot-resolution. Results can complement those from other analyses, especially those for determining low MW peptides. Notable effects were found for cheese composition and ripening temperature on gel pattern and on relative levels of selected proteolysis products. In both cheese varieties, most peptides reached a maximum during the first 3 ripening months and gradually disappeared as ripening advanced.     

Rheological Evaluation of Maturing Cheddar Cheese

A number of Cheddar cheese samples of different age, pH and moisture content have been examined rheologically and electrophoretically to determine whether the progressive changes in cheese texture were related to casein proteolysis. The force-compression curves obtained by crushing cubes of cheese between small flat plates at constant speed were different for the different cheese samples and were affected by the moisture content, pH and extent of α_sl-casein proteolysis that had taken place in the cheese. These results support a model of cheese micro structure in which an extensive network involving α_sl- casein molecules traverses the cheese and as the cheese ripens, chymosin cleavage of α_sl-casein weakens the protein network. Such a model explains the rapid decrease in Cheddar cheese yield-force that occurs during the early stages of ripening.     

Antioxidant activity of Cheddar cheeses at different stages of ripening

The aim of the study was to evaluate the changes in the antioxidant properties of Cheddar cheese at different stages of ripening using different assays: 2, 2'-azinobis (3 ethyl benzothiazoline)-6-sulphonic acid, 2, 2-diphenyl 1, picryl hydrazyl and superoxide radical scavenging activity. Cheddar cheese was prepared with Lactobacillus casei ssp. casei 300 and Lactobacillus paracasei ssp. paracasei 22 and without adjunct cultures. The antioxidant activity of water-soluble extracts of Cheddar cheese was dependent on the ripening period. The changes in the antioxidant activity were related to the rate of formation of soluble peptides (proteolysis) in all the samples of cheeses up to fourth month of ripening.     

Influence of probiotic bacteria on the proteolysis profile of a semi-hard cheese

Two probiotic strains, Lactobacillus acidophilus and Lactobacillus paracasei subsp. paracasei, were used as adjunct cultures in semi-hard cheesemaking experiments, in order to study their influence on proteolysis during ripening. Cheeses with and without probiotic bacteria were manufactured. The population of probiotics remained above 10⁷ cfu g⁻¹ during all ripening, and they did not influence primary proteolysis. However, L. acidophilus produced a significant increase in the level of low molecular weight nitrogen compounds and individual free amino acids; the amino acid profiles were also different. Multivariate analysis of peptide profiles showed that samples were grouped mainly by ripening time, although the impact of probiotics was also noticeable. L. acidophilus showed a clear influence on secondary proteolysis, while a minor effect of L. paracasei was evidenced at the end of the ripening. These results showed that the tested strains influenced distinctly proteolysis of cheeses, probably as a consequence of their different proteolytic systems and their activity via the alimentary matrix (cheese).     

Proteolytic activity of three probiotic strains in semi-hard cheese as single and mixed cultures: Lactobacillus acidophilus,Lactobacillus paracasei and Bifidobacterium lactis

The influence of three probiotic strains (Lactobacillus acidophilus, Lactobacillus paracasei and Bifidobacterium lactis) in semi-hard cheese proteolysis patterns was assessed. Probiotics were inoculated both as single cultures and as a three-strain mix, and added to milk either after a pre-incubation step or directly to the vat. B. lactis did not show any effect on proteolysis of cheeses, while L. paracasei showed limited impact at the end of the ripening. In contrast, L. acidophilus significantly influenced secondary proteolysis from the beginning of ripening, causing an increase in the levels of small nitrogen-containing compounds and free amino acids and changes in the peptide profiles. The effect of Lactobacillus acidophilus on peptidolysis was more noticeable when it was added to cheese–milk after pre-incubation in an enriched milk fat substrate. Similar results obtained with the three-strain mixed culture, suggesting that L. acidophilus played a major role in secondary proteolysis of probiotic cheeses in this trial.     

Angiotensin‐converting enzyme‐inhibitory and antithrombotic activities of soluble peptide extracts from buffalo and cow milk Cheddar cheeses

The aim of the study was to evaluate potential role of a water‐soluble peptide (WSP) extracts derived from buffalo and cow milk Cheddar cheeses with special reference to their antihypertension and antithrombotic activities. The WSP fractions collected at different stages of ripening were tested to assess their degree of proteolysis, their peptides were profiled by RP‐HPLC and in vitro assays for potential bioactivity were conducted. The peptide peak development was observed with slight differences in peaks number, area and height. Both angiotensin‐converting enzyme‐inhibitory and antithrombotic activities increased progressively during ripening. In comparison, the highest activities were observed in peptide extracts obtained from buffalo milk Cheddar cheese, in a dose‐dependent fashion.     

地衣芽孢杆菌凝乳酶对切达干酪成熟过程中蛋白水解的影响

利用地衣芽孢杆菌凝乳酶制作切达干酪和切达干酪类似物,分析干酪成熟过程中各蛋白水解指标的变化规律,以揭示地衣芽孢杆菌凝乳酶对切达干酪成熟过程中蛋白水解的影响。结果表明,CDF组（添加地衣芽孢杆菌D3.11凝乳酶所制切达干酪）、CD3组（添加地衣芽孢杆菌D3.11凝乳酶但未添加发酵剂制成的干酪类似物）和CCF组（添加商品凝乳酶所制切达干酪）干酪蛋白含量、pH 4.6-可溶性氮、12%三氯乙酸-可溶性氮、5%磷钨酸-可溶性氮、总游离氨基酸含量均随着成熟时间延长呈显著增加趋势,并且成熟期间CDF组干酪均显著高于CCF组干酪（P＜0.05）;十二烷基硫酸钠-聚丙烯酰氨凝胶电泳分析表明,CDF组干酪α-酪蛋白水解程度较大;pH 4.6-可溶性肽段分析表明,随着干酪的成熟,总肽含量呈先增加后下降趋势,但疏水性肽与亲水性肽的比值呈持续下降趋势,在成熟第6个月时,CDF组、CD3组和CCF组干酪疏水性肽与亲水性肽比值分别为2.668、2.822、3.788。主成分分析表明,3 组干酪的蛋白水解程度与成熟度呈正相关,与疏水性肽和亲水性肽的比值呈负相关。以上结果表明,利用地衣芽孢杆菌凝乳酶制作的干酪蛋白水解度更高,但其疏水性肽比例较小,研究结果可为地衣芽孢杆菌凝乳酶在干酪生产中的应用提供理论依据。     

瑞士乳杆菌对契达干酪成熟期间所产ACE抑制肽的影响及其消化稳定性

为明确瑞士乳杆菌对契达干酪中血管紧张素转换酶（angiotensin-converting enzyme,ACE）抑制肽活性的影响,以蛋白质水解度和ACE抑制率为指标,与干酪乳杆菌组、鼠李糖乳杆菌组和空白组干酪进行对照,研究瑞士乳杆菌对干酪成熟期间蛋白质水解及ACE抑制活性的影响,并对ACE抑制活性最高时期的干酪进行消化稳定性研究。结果表明：成熟期间,3 组益生菌干酪的活菌数无明显差异（P＞0.05）,但均高于空白组;益生菌干酪的蛋白质水解程度和ACE抑制活性显著高于空白组（P＜0.05）,其中瑞士乳杆菌干酪的蛋白质水解程度最强,活性最高（79.71%）。模拟消化后,瑞士乳杆菌干酪活菌数降低14.30%,ACE抑制活性显著增加（P＜0.05）,达到86.06%,多肽质量浓度增加至2.81 mg/mL;研究不同分子质量超滤组分消化后的ACE抑制活性发现,其中大于10 kDa的多肽活性升高,小于10 kDa的活性下降。此外,添加瑞士乳杆菌不影响干酪的整体可接受性。因此,瑞士乳杆菌能促进干酪ACE抑制肽的产生并提高其活性,消化后活性的升高主要与大分子肽的降解有关。