الفهرس 
IntroductionOnly 14 pages are availabe for public view
 |  | from | 306 |  |  |
| | | from | 306 | | |
Abstract
Sausage is considered one of the important widespread meat products in Egypt, that was manufactured in many butcher shops and sold fresh. Also, sausage was manufactured by small and main factories, that produced non-fermented (fresh) or fermented sausages. Many investigations confirmed that the sausage is one of the most important sources for human foodborne disease, where sausage and other meat products are considered a good media for many bacteria especially the pathogens. Therefore, this study was divided into three main parts to find out the optimum method for sausage preservation and to eliminate the pathogenic bacteria as follows:
1-Microbiological and chemical evaluation of the retailed sausage in Egyptian markets.
2-Study the antimicrobial activity of some essential oils and lactic acid bacteria against pathogenic bacteria.
3-Study the effect of some essential oils and lactic acid bacteria on microbiological changes, behaviour of pathogenic bacteria and their effect on chemical and organoleptic quality.
Part I. Microbiological and chemical evaluation of retailed sausage samples.
To study the microbiological and chemical quality of retailed sausage samples, 45 retailed commercial sausage samples were collected from different sources of Egyptian
markets (local markets of Qalubia, Cairo and Giza Governorates), which produced by butcher shops (fresh sausage), small and main factories (frozen and fermented sausages). Collected samples were microbiologically and chemically analyzed and pathogens detection was also carried out.
1- Microbiological quality. A- General microbiology.
1-Fermented sausage had the highest mean value of total microbial count, followed by fresh sausage. While, frozen sausage had the lowest mean value. Most of retailed sausage samples from the three types of sausage had higher total microbial counts than the limits allowed by Egyptian standards.
2-Fermented sausage had the minimum mean count of coliform bacteria, followed by fresh sausage then the frozen one which contained the higher level of coliform group. Most of sausage samples of each type except fermented type had coliform bacterial counts above the limits.
3-The highest count of lactic acid bacteria was observed in fermented sausage type, followed by frozen sausage, while fresh sausage recorded the minimum average level of lactic acid bacteria.
4-Yeasts and moulds counts of fresh, frozen and fermented sausage samples indicated that, fermented sausage had the highest mean value of yeasts and moulds followed by frozen and fresh sausages.
5-Proteolytic bacterial count of fermented sausage recorded the highest mean value of proteolytic bacteria followed by fresh sausage, while the frozen type showed the lowest mean value.
6-Fermented sausage had the highest mean level of lipolytic bacterial count, while the numbers were almostly similar for frozen and fresh sausages.
B- Pathogens detection in retailed sausage samples.
1-All retailed sausage samples from three types of sausage were positive for the presence of Staphylococcus aureus, where Staphylococcus aureus were present in 100% of samples. Fresh sausage had the highest mean count of S. aureus, while fermented and frozen had the lowest one.
2-Results indicated that Listeria monocytogens was present at higher percentage, represented 86.66%, 80% and 73.33% for fresh, frozen and fermented sausage, respectively. Fermented sausage had the lowest percent of Listeria monocytogens occurrence.
3-Results indicated that, E. coli 0157:H7 was present in commercial sausage samples at rates of 60%, 53.33% and 46.66% for fresh, frozen and fermented sausage samples, respectively.
4-The lowest percentage of Salmonella spp detection observed with fermented sausage (20%), followed frozen sausage (40%). While fresh sausage had the highest percent of Salmonella sp occurrence (46.66%).
2- Chemical quality of retailed sausage samples.
1-pH values of retailed sausage samples were highly varied among samples and from sausage type to another. Fermented sausage recorded the lowest pH mean value (4.85), while both fresh and frozen sausage had pH mean values 6.52 and 6.51, respectively.
2-Fresh sausage recorded the lowest mean value of salt content (2.87%), while frozen and fermented sausages contained 3.45% and 3.42%, respectively.
3-Sodium nitrite was not added to butcher sausage samples (fresh) except for 3 of 15 samples (20%) with average level 266.5 ppm. While frozen sausage had higher sodium nitrite content with average level 223.03 ppm. whereas, fermented sausage had lowest average level 49.16 ppm. Sodium nitrite was used at high levels which exceeded those of Egyptian standard specifications in 100% of frozen sausage samples (15 of 15) and in 100% of butcher sausage samples (fresh). In addition, only fermented sausage contained sodium nitrite at permissible limits.
4-The lowest total volatile nitrogen (TVN) values were observed in fresh sausage, followed by fermented one, while frozen samples had the highest TVN values than other two types.
5-Fresh sausage had the lowest values of thiobarbaturic acid (TBA), followed by frozen sausage, while, the highest mean value of TBA was observed in fermented sausage samples
Part II. Antimicrobial activity of some essential oils and lactic acid bacteria against pathogenic bacteria.
1. Antimicrobial activity of spices essential oils:
Preliminary screening of antimicrobial activity of 8 essential oils (clove, black cumin, garlic, onion, cumin, anise, mint and marjoram) against pathogenic bacteria was achieved. Results indicated the following:
1-All essential oils inhibited strain of Staphylococcus aureus. The maximum activity was for mint, marjoram and cumin, while the minimum activity was for garlic and clove essential oils.
2-All essential oils inhibited Listeria monocytogens strain. Cumin essential oil, followed by mint, and marjoram essential oils showed the highest antimicrobial activity against Listeria monocytogens. While both garlic and onion essential oils showed the lowest one.
3-Strain of E. coli 0157:117 was inhibited with all tested essential oils. Marjoram followed by onion and cumin essential oils had the highest antimicrobial activity, then mint essential oil. While, garlic essential oil showed the lowest antimicrobial activity.
4-Salmonella sp also inhibited with all tested essential oils. Cumin essential oil had the highest antimicrobial activity, followed by marjoram and mint essential oils. While, the minimum inhibition action was recorded by anise essential oil.
It could be concluded that the maximum mean values of inhibition zones against the tested pathogenic bacteria were obtained by marjoram> cumin > mint. Followed by black cumin, onion and anise.
5- Minimum inhibitory concentration for selected essential oils (marjoram, cumin and mint) showed that both S. aureus and Listeria monocytogenes were inhibited at 500ppm with all tested essential oils (marjoram, cumin and mint). While, E. coli 0157:H7 was inhibited at 3000ppm with all essential oils, Salmonella sp was inhibited at 1500, 1500 and 2000ppm of cumin, mint and marjoram essential oils, respectively.
2- Antimicrobial activity of lactic acid bacteria.
All pathogenic strains were inhibited by both Lactococcus Lactis or Lactobacillus plantarum and its mixture. The three treatments recorded inhibition zone 8.5mm, 9.5mm and 10.5mm against Staphylococcus aureus, respectively, while it was 1 Omm, 12.25mm and 11.5mm against Listeria monocytogenes, also inhibition zone diameter was 9.5mm, 12mm and 14mm against E. coil and was 9.5mm, 10.5mm and 9mm against Salmonella sp, respectively.
Part III. Effect of different treatments on microbiological and chemical changes of
experimental sausage during storage.
In this part of study each of strain of S. aureus, Listeria monocytogenes, E. coli 0157: H7 and Salmonella sp were individually added to sausage mixture. The first sausage type
(non-fermented) treated with either cumin or marjoram essential oils individually added against each of pathogen. In the second type (fermented sausage), starter of Lactococcus lactis and L. plantarum was added against each pathogen. After processing each sausage type, various treatments were cold stored at 4-5°C up to 60 days. The microbiological, chemical and organoleptic evaluations were carried out initially and at 3, 7, 15, 30 and 60 days.
1. Microbiological quality.
A. Changes in microbial populations.
1-Total microbial counts of sausages treated with essential oils were gradually increased with increasing period of storage up to 60 days. Sausage mixture treatments inoculated with various pathogens (positive control) recorded higher total microbial counts than negative control (non-inoculated one), while treatments contained either cumin or marjoram essential oils showed the minimum increment level during all storage periods.
Total microbial counts of fermented sausage increased by extending the storage period, but the highest increase rate was shown after fermentation, then slight increase was shown. Total microbial counts of starter treatments with S. aureus or with Listeria monocytogens decreased at 3 days. The increment rate was lower in starter treatments, than other fermented treatments.
2-A fair decrease was noticed in coliform bacterial counts of all sausage treatments by increasing the storage period.
The highest decrement rate was shown in both cumin or marjoram treatments, where coliform bacteria completely disappeared at 15 days for all cumin or marjoram treatments. While coliform bacteria disappeared at 60 days for all control treatments except for both E. coli 0157: H7 and Salmonella sp treatments.
On the other hand a marked decrease in coliform bacterial counts was shown in all treatments of fermented sausage after fermentation. The faster decrement was noticed in starter treatments than that of both negative control and positive controls. Coliform bacteria were disappeared at 7 days for all starter treatments.
3-Lactic acid bacterial counts of non-fermented sausage gradually increased with increasing storage period, then decreased in later stages of storage. All cumin treatments recorded the lower increase rate of lactic acid bacteria, followed by marjoram treatments compared with negative control and positive controls.
Lactic acid bacterial counts of fermented sausage highly increased immediately after fermentation. Starter addition treatments showed the maximum lactic acid bacterial counts at all intervals of storage period.
4-Yeasts and moulds counts slightly increased at the beginning of storage period, followed by gradual increase in all treatments of non-fermented sausage. Cumin treatments recorded the lowest increase during storage, followed by marjoram treatments.
On the other hand, yeasts and moulds counts remained low at the end of fermentation (at 3 days) when the starter bacterial counts progressed, but their numbers rapidly increased by increasing the storage period. Starter treatments showed higher yeasts and moulds counts compared to uninoculated treatments.
5-Proteolytic bacterial counts of non-fermented sausage treatments increased by increasing the storage period. The proteolytic bacterial counts of both cumin and marjoram treatments were lower than control treatments.
Also, proteolytic bacterial counts of fermented sausages treatments increased by prolonging the storage period up to 60 days. Starter treatments had a slight higher proteolytic bacterial count than controls. On the other hand, fermented sausages had higher proteolytic bacterial counts than that of essential oils treated one.
6-Positive control treatments showed lipolytic bacterial counts higher than that of negative control at almost all periods. While, cumin followed by marjoram added treatments showed the lowest lipolytic bacterial counts and the lowest increase ratio than other treatments.
On the other hand, lipolytic bacterial counts of fermented sausages followed the same trend of essential oils treated sausages, but the fermented sausages had higher numbers of lipotyitc bacteria than that of non-fermented sausages (essential oils treated sausages). Starter treatments had the highest lipolytic bacterial counts at almost all storage periods.
B- Survival of pathogenic bacteria.
Pathogenic strains isolated from retailed sausage samples were added to experimental preparing sausage to evaluate the effect of different treatments on pathogens survival.
1-S. aureus counts decreased at the 3rd day of cold storage, then increased by increasing storage period in negative control treatment. While, in S. aureus treatments, the counts decreased from 4.92 and 4.95 logio/g at initial time to 2.94 logidg and 2.99 logio/g at the 3rd day of storage for cumin and marjoram treatments, respectively. The high decrease was observed in cumin followed by marjoram treatments.
Also, S. aureus counts highly decreased in S. aureus added fermented treatments immediately after fermentation. Starter treatment showed the greatest decrease of S. aureus counts.
2-Negative control treatment of non-fermented sausage was free from Listeria sp. While, in Listeria monocytogenes added treatments, the counts decreased at 3days, then the counts increased in positive control treatment by increasing the storage period. Continuous decrease was observed either with cumin or marjoram added treatments up to 60 days.
Also, Listeria sp was not detected in fermented negative control treatment. Positive control treatment showed slight decrease in the numbers of L. monocytogens immediately after fermentation, then increased by
increasing the storage period. Starter treatment showed highest reduction level and lowest L. monocytogenes counts.
3-On the other hand, positive control of E. coil 0157: H7 showed a slight decrease in counts, then increased with increasing the storage period. While, both of marjoram followed by cumin E. coil treatments recorded highest reduction level and the minimum counts.
In fermented sausage treatments E. coil 0157:H7 was not found in negative control treatment. Positive control showed a slight decrease in the number of pathogens immediately after fermentation, then increased by increasing the storage period. While addition starter to E. coli treatment showed a great reduction in the pathogen numbers. E. coil 0157: H7 didn’t disappear during 60 days of cold storage from both positive control and starter added treatments.
4-Also, positive control of Salmonella sp showed a slight decrease in counts at 3 days, then increased with increasing the storage period. While, treatment of marjoram followed by cumin with Salmonella sp recorded highest reduction level and the minimum counts.
Salmonella sp was not detected in fermented negative control treatment. While, in positive control a slight decrease in the number of pathogens was observed at 3 days, then the numbers were increased by increasing the storage period. Starter addition to Salmonella sp treatment showed a major reduction in the pathogen numbers.
2-Chemical changes of experimental sausage samples during 60 days of cold storage (4-5°C).
1 All positive control treatments of non-fermented sausage showed gradual increase in pH values at all stages of storage. On the other hand, both cumin or marjoram treatments showed constant pH values at the earlier stages of storage, then pH values were gradually increased with increasing the storage period. Cumin or marjoram treatments showed the lowest increment rate and the lowest pH values during all storage intervals.
On contrast, pH values of all fermented treatments highly decreased by fermentation to reach minimum, then slightly increased up to the end of storage period. Starter added treatments showed the lowest pH values.
2-Salt content of all experimental sausage treatments ranged between 2.73% to 2.88% at initial time. Salt content increased by increasing the storage period. There are no differences between all treatments (fermented and non-fermented) concerning with changes in salt content during storage.
3-Nitrite content decreased by increasing the storage period for all non-fermented treatments. Both cumin or marjoram treatments showed lower nitrite content and maximum nitrite reduction level.
Also, nitrite content of fermented sausage treatments decreased by increasing the cold storage period for all fermented treatments and reached minimum values
at the end of storage period. Starter added treatments with various pathogens recorded the rapid decrement and the lowest levels than both negative control and positive controls.
4- Total volatile nitrogen of essential oils treated sausages of various treatments ranged between 8.1 to 11.9 mg/100gm at the initial time. Its concentration was gradually increase by prolonging storage period for all treatments. Both cumin or marjoram treatments showed the lowest increment ratio and lowest levels of total volatile nitrogen up to the end of storage.
On the other side, total volatile nitrogen of fermented sausage treatments increased by increasing the storage period, with lower levels than that of essential oils treated sausages. Starter treatments showed an increase in total volatile nitrogen by increasing the storage period, but the increment rate was lower than that of negative control and positive controls.
5- Thiobarbaturic acid (TBA) values of non-fermented sausage treatments were gradually increased with extending the storage period, reaching the maximum at the end of storage (60 days). Cumin or marjoram treatments recorded the lower increase rate and the lowest TBA values during all period intervals than negative control and positive control treatments.
Also, thiobarbaturic acid values of fermented sausage treatments increased by increasing storage period and reached maximum at the end of storage. While, TBA
of starter treatments slightly increased by increasing the storage period. All starter treatments recorded lower TBA levels than both negative control and positive controls at all period intervals.
4- Changes of organ oleptic properties.
All essential oils treatments were in the ”very good” grade (20-24) at the beginning of storage. These scores slightly decreased at the earlier stages of storage, then gradual decrease was observed. Cumin followed by marjoram treatments recorded the lowest decrement rate in overall scores than that of negative control and positive controls. While positive controls treatments showed the highest reduction in overall scores, followed by negative control treatment. Cumin treatment was the more constant treatment for organoleptic properties, followed by negative control and marjoram treatments.
On the other hand, organoleptic evaluation of fermented sausage treatments increased immediately after fermentation and after storage to reach maximum, then slight decrease was observed. All fermented sausage treatments were ”very good” grade up to 15 days. Starter added treatments showed maximum organoleptic scores compared to negative control and positive controls treatments.