Isolation, Identification and Selection of Lactic Acid Bacteria Cultures from Mum (Thai Fermented Sausages) and Their Applications

Mum，為一種傳統的泰式發酵香腸，普遍生產並消費於泰國東北部地區，但在生產過程中，卻少有科學化和技術被應用於其中。傳統上，mum香腸是利用天然乳酸菌，因此也易導致產品品質的不穩定。製造商常依生產技術和經驗而並非科技以維持產品質。所以影響產品的最終特性與品質主要來自原物料品質及生產技術。因此，本研究的目的是藉由一種新的方法以改善mum香腸之品質及保存期限並利用乳 酸菌菌酛的接種以建立一個更穩定並可控制的加工程序。 在試驗一中，兩種不同方法用於生產新鮮mum香腸，方法一：香腸於30°C下進行發酵和儲存14天;方法二：香腸在30℃下發酵三天，再以真空包裝存儲於4°C，直到第28天。於過程中取樣測定其物理特性、微生物，質地，感官評品。結果顯示，方法一的樣本之脫水現象較方法二之樣品為明顯並導致較低的水分含量和水活性。在第3天時，這兩方法之樣品其pH值均有顯著下降但乳酸則有顯著增加。總生菌數及乳酸菌數在發酵過程中，菌數先是迅速升高，然後下降，而腸內桿菌則是持續下降。方法一之樣品則因脫水會造成質地堅硬和不可接受的感官品質。綜上所述，方法二之樣品於三天的發酵後使用真空包裝並儲存在4°C下，更能生產 較佳品質且保存期限較長的mum香腸。 在試驗二中，從試驗一中之2種方法製造的mum香腸進行乳酸菌的分離，再將分離出的乳酸菌(LAB)其16S rDNA 利用聚合酶鏈反應擴增，並用限制內切酶進行消化。結果顯示，Dde I 最具有辨別能力。乳酸菌於更進一步分類後共計有Lactobacillus sakei, Lactobacillus plantarum, Leuconostoc mesenteroides, Lactobacillus brevis, Lactobacillus fermentum, Pediococcus pentosaceus, 和 Lactococcus lactis。 在這兩種製造方法中L. sakei 和 L. plantarum 在發酵和儲存過程中是為優勢菌種。而方法二中，Ln. mesenteroides 的比例於儲存熟成期間很明顯增加且類似於 L. sakei是為次優勢菌。樣品中的乳酸菌的鑑定將有助 於選擇適當的菌酛以進一步使用。 在試驗三中，添加混合發酵菌酛（L.plantarum，L.sakei和Lc.lactis）及亞硝酸鈉（125 ppm）是可提高mum香腸之產品品質和安全性。結果顯示不僅可顯著降低mum香腸之pH值及抑制腸內桿菌生長，亦可抑制TBARS及VBN的積累；同時有減少亞硝酸鹽的殘存量，及提高紅色值的效果。另外，使用混合發酵菌酛與亞硝酸鹽所生產之mum香腸，其各項感官評品尤其是風味，顏色和總接受性均有最高的得分。 綜上所述，利用真空包裝、混合發酵菌酛和亞硝酸鹽的組合，證實能提高mum香腸之品質及安全性並延長其保存期。Mum, which is a traditional Thai fermented sausage, is commonly produced and consumed in the north-eastern region of Thailand, and yet little scientific and technology information is applied in production. Traditionally, mum sausage is produced by utilizing natural occurring lactic acid bacteria (LAB), and it may cause the products with inconsistent qualities. Their production depends on the skill and experience of the meat manufacturers rather than technology. Hence, the specific characteristics and qualities of final products are mainly influenced by the raw materials source, the manufacturing techniques and the agro-ecosystem of the production area. Thus, the aim of this study was to improve the qualities and the shelf-life of mum sausages by a new method and also to develop desirable LAB starters in order to establish a more controllable processing. In experiment I, freshly-manufactured mum sausages were produced by two processing methods (process I: fermented and stored at 30�C for 14 days; process II: fermented at 30�C for three days, vacuum-packaged and stored at 4�C until day 28). Physiochemical, microbial, textural, and sensory properties of the sausages were analyzed. The results showed that dehydration was more extensive in process I samples, and resulted in lower moisture content and water activity than process II samples. Significant decreases in pH values and increases in lactic acid were observed in both samples at day 3. The total microflora and lactic acid bacteria counts increased rapidly during the fermentation and then decreased as Enterobacteriaceae counts decreased. Extensive dehydration resulted in tough textures and unacceptable sensory qualities for the process I samples. In conclusion, three days of fermentation, with vacuum-packaging, ripening and storage at 4�C, were possibly to produce mum sausages with better qualities and more extended shelf life. In experiment II, LAB were isolated from the sausages during manufacturing by two methods (method I: after stuffing, sausages were fermented and stored at 30�C for 14 days; method II: after stuffing, sausages were fermented at 30�C for three days, then were vacuum-packaged and stored at 4�C until day 28). The 16S rDNA of LAB isolate was amplified by polymerase chain reactions and digested with restriction enzymes. The results showed that Dde I presented the highest discrimination capacity. LAB were classified and further identified as Lactobacillus sakei, Lactobacillus plantarum, Leuconostoc mesenteroides, Lactobacillus brevis, Lactobacillus fermentum, Pediococcus pentosaceus, and Lactococcus lactis. During fermentation and storage, L. sakei and L. plantarum were dominant in both methods. For method II, the proportion of Ln. mesenteroides increased remarkably during post-ripening storage and became predominant similar to L. sakei. The identification of LAB in samples helps to select appropriate microorganisms as candidate starter cultures for future use. In experiment III, the addition of selected mixed starter culture (L. plantarum, L. sakei, and Lc. lactis) with NaNO2 (125 ppm) had a great potential to improve quality and safety of mum sausages, which showed a significant decrease in pH values, inhibiting the growth of Enterobacteriaceae, and suppressing the accumulation of TBARS and VBN. Also, it showed an ability to reduce nitrite content and revealed a higher redness values. Moreover, sausages produced with mixed starter culture and nitrite had the highest scores in most sensory attributes especially in flavor, color, and overall acceptance. Thus, the combination of several hurdles including vacuum-packaging, mixed starter culture and additive in the manufacture of mum product evidenced that can improve its qualities, safety and extend the shelf-life.TABLE OF CONTENTS Page ACKNOWLEDGMENTS i ABSTRACT ii TABLE OF CONTENTS vi LIST OF TABLES viii LIST OF FIGURES ix CHAPTER 1: Introduction……………………………………………………………………..… 1 1. Rationale and background ………………………………………………... 2 2. Aim of this study………………………..…………...…………………….. 4 CHAPTER 2: Literature review……………………………………………………………….…. 5 1. Fermentation of meat…………………………………………………….… 6 2. Lactic acid bacteria………………………………………………………… 8 3. Identification of lactic acid bacteria……………………………………….. 12 4. Role of lactic acid bacteria in food preservation ………………………..… 18 5. Lactic acid bacteria as functional starter cultures…………………….…… 22 6. Lactic acid bacteria as probiotics ……………………………………….… 26 CHAPTER 3: An innovative method for the mum (Thai fermented sausages) preparation with acceptable technological quality and extended shelf-life…………………….…... 28 1. Abstract……………………………………………………….…………..... 29 2. Introduction…………………………………………………………...…… 29 3. Materials and methods……………………………………………..………. 31 4. Results and discussion………………………….………………………….. 33 Page CHAPTER 4: Monitoring the dynamics of lactic acid bacteria population during manufacturing and storage of mum (Thai fermented sausage) based on restriction fragment length polymorphism (RFLP) analysis…………………………………………… 47 1. Abstract…………………………………………….….……….………....... 48 2. Introduction………………………………………………………………... 48 3. Materials and methods…………………………………………….……….. 50 4. Results and discussion……………………………………………………... 52 CHAPTER 5: The addition of starter cultures and nitrite to improve safety and quality of mum (Thai fermented sausages)………………………………………………………... 62 1. Abstract……………………………………………………….………......... 63 2. Introduction………………………………………………………………... 63 3. Materials and methods………………………………………………..……. 65 4. Results and discussion……………………………………………….…….. 68 CHAPTER 6: Conclusion………………………………………………………………………... 87 REFERENCES………………………………………………………………….... 90 APPENDICES……………………………………………………………………. 11

An innovative method for the preparation of mum (Thai fermented sausages) with acceptable technological quality and extended shelf-life

Freshly-manufactured mum sausages were assigned to two processing methods (process I: stored at 30 C for 14 days; process II: stored at 30 C for three days, vacuum-packaged, and stored at 4 C until day 28). Physicochemical, microbial, textural, and sensory properties of samples were analysed. The results showed that dehydration was more intense in process I samples, and resulted in lower moisture content and water activity. Significant decreases in pH values, and increases in lactic acid were observed in both samples by day 3. The total microflora and lactic acid bacteria counts increased rapidly during the fermentation and then decreased while the Enterobacteriaceae counts decreased steadily. Too much dehydration resulted in tough textures and unacceptable sensory qualities for process I samples. In conclusion, after three days of fermentation, with vacuum-packaging, ripening and storage at 4 C up to 28 days, it is possible to produce mum sausages with better qualities and an extended shelf life