Ong, Lydia (2007) Influence of probiotic organisms on proteolytic pattern, release of bioactive compounds and sensory attributes of cheddar cheese. PhD thesis, Victoria University.

Abstract

The overall objective was to study the influence of probiotic organisms on the proteolytic pattern, sensory evaluation and release of bioactive peptides in Cheddar cheeses. The study consists of three main parts. The first part of the study aimed at development of Cheddar cheeses with the addition of probiotic organisms. Three batches of Cheddar cheeses including a control cheese with starter lactococci only (Batch 1M) and two probiotic cheeses with starter lactococci and probiotic organisms including Bifidobacterium longum 1941, Lactobacillus casei 279 and Lb. acidophilus 4962 (Batch 2M) or B. animalis subsp. lactis LAFTI®B94, Lb. casei LAFTI®L26 and Lb. acidophilus LAFTI®L10 (Batch 3M) were made. In a separate experiment, seven batches of Cheddar cheeses including a control cheese with starter lactococci only (Batch 1) and six probiotic cheeses with starter lactococci and probiotic B. longum 1941 (Batch 2), B. animalis subsp. lactis LAFTI®B94 (Batch 3), Lb. casei 279 (Batch 4), Lb. casei LAFTI®L26 (Batch 5), Lb. acidophilus 4962 (Batch 6) or Lb. acidophilus LAFTI®L10 (Batch 7) were made. All cheeses were made in triplicate and ripened at 4°C for 24 wk to study the influence of the probiotic adjuncts on proteolytic patterns and organic acid profiles of the cheeses. The probiotic bacteria were added as an adjunct together with the starter lactococci and no alteration in the cheese-making procedure was necessary for incorporation of probiotic organisms into Cheddar cheese. The probiotic adjuncts survived at high levels at the end of cheese-making process (8.0 - 9.0 log10 cfu g-1). They were also able to maintain viability at > 7.5 log10 cfu g-1 at the end of ripening at 4oC for 24 wk. The counts of lactococci in the cheeses decreased by one to two logs at the end of ripening, but counts were not significantly different (P > 0.05) between the control and probiotic cheeses. Addition of probiotic organisms did not alter the composition of cheeses (fat, protein, moisture, salt contents), but acetic acid concentration in probiotic cheeses (Batches 2M & 3M) was significantly higher than that of the control cheese (Batch 1M). Assessment of proteolysis during ripening showed that the concentrations of trichloroacetic acid-soluble nitrogen (TCA-SN) and phosphotungstic acid-soluble nitrogen (PTA-SN) in probiotic cheeses (Batches 2M & 3M) were significantly higher (P < 0.05) than those of the control cheeses (Batch 1M). Hydrolysis of αs1-casein (CN) in the cheeses was 19.28, 46.99 and 63.42 after 24 wk of ripening in Batch 1M, Batch 2M and Batch 3M, respectively. The results show that Cheddar cheeses can be an effective vehicle for delivery of probiotic organisms to the consumer. Each probiotic organisms influenced the organic acid profiles and the proteolytic pattern of Cheddar cheese in different ways. Acetic acid concentration in probiotic cheeses with B. longum 1941, B. animalis subsp. lactis LAFTI®B94, Lb. casei 279 or Lb. casei LAFTI®L26 (Batches 2-5) was higher as compared to other cheeses. Cheeses made with the addition of Lb. casei 279 (Batch 4) and Lb. casei LAFTI®L26 (Batch 5) showed higher level of αs1-CN and β-CN hydrolysis when compared to other cheeses. Although Bifidobacterium sp. was found to be weakly proteolytic, cheeses with the addition of that species had the highest concentration of PTA-SN. The sensory properties of the probiotic Cheddar cheeses were assessed after ripening for 9 months at 4oC. Probiotic cheeses except those with Lb. acidophilus 4962 (Batch 6) were found to be significantly different (P < 0.05) from the control cheeses made without any probiotic organism (Batch 1). The acceptability scores among the cheeses, however, were not significantly different except for that with Lb. casei 279 (Batch 4). Acceptability scores of cheese with Lb. casei 279 (Batch 4) was significantly lower (P < 0.05) than that of the control cheese with bitterness and sour-acid taste as the major defects. Although concentration of acetic acid in probiotic cheeses was higher than the control cheese, there was no significant correlation between the sensory scores of vinegary and the acetic acid concentration (P > 0.05). Scores of vinegary also did not influence the acceptability of the cheeses (P > 0.05). Increase proteolysis in probiotic cheeses did not influence the scores of Cheddary attribute (P > 0.05). There were positive correlations (P < 0.05) between the scores of bitterness and the level of water-soluble nitrogen. The sensory results showed that some of the probiotic microorganims used in this study can be applied successfully in Cheddar cheeses with acceptable sensory profiles. The increase in proteolysis in cheeses with the addition of probiotic organisms indicated that more peptides were released into the probiotic cheeses. The angiotensin converting enzyme (ACE)-inhibitory activity of the water soluble extract (WSE) of cheeses was determined during ripening at 4oC. The IC50 (concentration of ACE-inhibitory peptides needed to inhibit 50% of ACE activity) was the lowest after 24 wk of ripening in the probiotic cheeses (0.20 - 0.29 mg mL-1) compared to 36 wk for cheeses without any probiotic (0.28 – 0.31 mg mL-1). Cheeses made with Lb. casei 279 (Batch 4) or Lb. casei LAFTI®L26 (Batch 5) with the highest degree of proteolysis and a control cheese (Batch 1) were selected for isolation and purification of bioactive peptides. Water soluble extracts of each cheese were subjected to several stages of chromatographic fractionation. Inhibitory activity found in the crude fractions ranged from 0.1 to 2.0 mg mL-1. Fractions with the highest activity were purified using a second stage chromatography. Various ACE-inhibitory peptides corresponding to the αs1-casein [(f 1-6), (f 1-7), (f 1-9), (f 24-32) and (f 102-110)] and β-casein [(f 47-52) and (f 193-209)] were identified. Our results suggested that ACE inhibition in Cheddar cheeses was dependent on proteolysis to a certain extent. Probiotics Lb. casei 279 or Lb. casei LAFTI®L26 used in this study have the potential to improve the ACE-inhibitory activity of Cheddar cheeses. The second part of the study investigated the influence of ripening temperatures at 4 and 8oC on the viability of probiotic organisms, composition of cheeses, production of organic acids, proteolytic pattern, sensory characteristics and ACE-inhibitory activity of Cheddar cheeses. Seven batches of Cheddar cheeses were made in triplicate as in part I (Batches 1-7). The cheeses were divided into two equal portions and assigned to ripening at 4 and 8oC for 24 wk. The moisture content and pH of cheeses decreased significantly after ripening for 24 wk, depending on the probiotic adjuncts and ripening temperature used (P < 0.05). Ripening at 8oC accelerated the loss of starter lactococci as compared to 4oC. The counts of starter lactococci in cheeses produced with B. animalis LAFTI®B94 subsp. lactis, Lb. casei LAFTI®L26 or Lb. acidophilus 4962 ripened at 8ºC were significantly lower than that ripened at 4ºC (P < 0.05) at 24 wk. Probiotic organisms remained viable at the end of 24 wk and their viability was not affected by the ripening temperatures (P > 0.05). There were significant effects of the type of probiotic organisms used, ripening time, ripening temperatures and their interactions on the concentration of lactic, and acetic acids in the cheeses (P < 0.05). Lactic acid concentration was the highest in cheeses with Lb. casei 279 or Lb. casei LAFTI®L26 ripened at 8oC. The acetic acid concentration in cheeses made with Bifidobacterium sp. or Lb. casei sp. was significantly higher than that of the control cheese (P < 0.05). Citric, propionic and succinic acids contents of the cheeses were not significantly affected by the type of probiotic organisms or ripening temperatures used (P > 0.05). Ripening at 8oC as compared to 4oC also increased the level of proteolysis of the cheeses. Product of proteolysis and organic acids released during ripening were shown to be important for the flavour of Cheddar cheeses. There were positive and significant correlations between the levels of soluble nitrogen, lactic, acetic and butyric acids, percentage hydrolysis of αs1-CN and β-CN to the scores of cheddary flavour (P < 0.05). Scores for sour-acid and vinegary were higher in cheeses with the addition of Bifidobacterium sp. or Lb. casei 279 ripened at 8oC. The scores were positively and significantly correlated to the level of lactic, acetic and free amino acids in the cheeses (P < 0.05). ACE-inhibitory activity of the cheeses ripened at 4 and 8oC was maximum at 24 wk and remained relatively constant after that period. Cheeses made with the addition of Lb. casei 279, Lb. casei LAFTI®L26 or Lb. acidophilus LAFTI®L10 had significantly higher (P < 0.05) ACE-inhibitory activity than those without any probiotic adjunct after 24 wk at 4 and 8oC. The IC50 of cheeses ripened at 4°C was not significantly different (P > 0.05) to that ripened at 8°C. The lowest value of the IC50 (0.13 mg mL-1) and therefore the highest ACEinhibitory activity corresponded to the cheese made with the addition of Lb. acidophilus LAFTI®L10 ripened at 8°C. Several ACE-inhibitory peptides from WSE of cheeses with Lb. acidophilus LAFTI®L10 were isolated and identified as κ-CN (f 96-102), αs1-CN (f 1- 9), αs1-CN (f 1-7), αs1-CN (f 1-6), αs1-CN (f 24-32) and b-CN (f 193-209). Most of the ACE-inhibitory peptides accumulated during ripening, and as proteolysis proceeded, some of the peptides were hydrolyzed into smaller peptides. The results of the second part of the study showed that both 4 and 8oC can be used for ripening of probiotic Cheddar cheeses. Cheeses made with Lb. acidophilus LAFTI®L10 also had good potential for the development of Cheddar cheeses with bioactive properties. Cheeses made with Lb. acidophilus LAFTI®L10, however, was not the cheeses with the highest level of proteolysis. Part 3 of the study investigated the use of elevated ripening temperature of 12oC and the addition of a highly proteolytic strain of Lb. helveticus to improve the proteolysis and the ACE-inhibitory activity of probiotic cheeses made with Lb. acidophilus LAFTI®L10 adjunct. Cheddar cheeses were made with starter lactococci (control), Lb. acidophilus LAFTI®L10 and starter lactococci (L10) or Lb. acidophilus LAFTI®L10, Lb. helveticus H100 and starter lactococci (H100). The counts of probiotic organisms in L10 cheeses remained at >106 cfu g-1 after 24 wk of ripening at 4, 8 and 12°C. Concentrations of lactic, acetic and propionic acids of the L10 and H100 cheeses were significantly higher than those of the control cheeses after 24 wk of ripening (P < 0.05). Proteolysis of the cheeses improved as the ripening temperature increased. WSN, TCA-SN and PTA-SN of L10 and H100 cheeses were significantly higher than those of the control cheeses (P < 0.05). Increase in ripening temperature from 4oC to 8 and 12oC increased the percentage of ACEinhibition. The IC50 value among cheeses ripened at 4, 8 and 12°C, however, was not significantly different (P > 0.05). Addition of Lb. helveticus H100 did not further improve the proteolysis and the ACE-inhibitory activity of the probiotic cheeses. Overall results of the study showed that some probiotic organisms used in this study can be added successfully in Cheddar cheeses with acceptable organic acid, proteolysis and sensory profiles. Addition of probiotic Lb. casei 279, Lb. casei LAFTI®L26 and Lb. acidophilus LAFTI®L10 has the potential to improve the ACE-inhibitory activity of Cheddar cheeses.

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