Introduction

Lactic acid bacteria (LAB) are a group of probiotics that are generally recognized as safe by  the American Food and Drug Administration (FDA). These bacteria play a crucial role in the  fermentation of dairy products and are commonly found in various foods. LAB are  extensively researched for their probiotic qualities, which include enhancing gut health,  boosting the immune system, and inhibiting the growth of pathogenic bacteria. Due to these  health benefits, there is a growing interest in identifying new and alternative sources of LAB.  Traditionally, LAB have been found from common dairy products, but recent research has  expanded to isolating LAB from unconventional sources. Studies have successfully identified  LAB from oregano leaves, sugarcane, papaya, taro, and yam leaves, highlighting the  diverse environments in which these beneficial bacteria can thrive. This broadening of  research scope aims to discover new strains of LAB with unique probiotic properties and  potential applications in food and health industries. One study in particular involves the  isolation of Lactobacilli, a prominent LAB used in milk fermentation to produce yogurt, from  the stalks or calyx of chili plants. Although chili stalks are typically considered waste in food  processing, this discovery suggests that they could serve as a valuable source of LAB. The presence of Lactobacilli in chili stalks indicates their potential to curdle milk and create  yogurt, transforming what was once a by-product into a resource with significant economic  value.  

This research repositions chili stalks from being a waste product to a resource of high  potential. By utilizing the LAB found in chili stalks, there is an opportunity to enhance sustainable food production and develop value-added products. This study aims to explore the viability of using LAB from chili stalks for milk fermentation, thereby contributing to the diversification of probiotic sources and promoting the efficient use of agricultural by-products. The successful application of LAB from chili stalks could lead to innovative approaches in  food biotechnology and the dairy industry, ultimately supporting sustainability and economic growth.

Previous studies

The previous study introduced the lactobacilli to the milk by placing the sterilized chili stalks immediately into the milk. Their results suggested that the chili stalks were able to lower the pH to the expected pH range of yogurt, however had a higher titratable acidity than expected.

Isolation of Bacteria from Chili Stalks

Sterilization:  The chili stalks were sterilized with Hydrogen Peroxide (H2O2) for 2 minutes, rinsed using 70% of  Ethanol for 5 minutes, treated repeatedly using 3% sodium hypochlorite twice.  Isolation:  A chili stalk was plated on selective media, i.e. De Man, Rogosa and Sharpe (MRS) agar  to  cultivate its bacteria. The zigzag and four-quadrant streak methods were then used to isolate a  single bacterial colony.  

Notes:  

• Bacteria was transferred using an inoculation loop from each petri dish to another. 

• Each Petri dish was incubated at 37°C for 2-3 days before proceeding to the next step of  bacterial isolation.

Identification of Bacteria (Gram Staining)

A single bacterial colony from the four-quadrant streak method was transferred to an MRS broth  using an inoculation loop. The broth was incubated for 2-3 days, after which an inoculation loop  was used to transfer a small amount of bacteria to a water drop on a glass slide.  

The glass slide was then heated using a bunsen burner to heat-fix the bacteria. Crystal violet was applied as the primary stain, followed by Lugol's iodine as a mordant. Acetone-alcohol was used  for decolorization, and safranin served as the counterstain. Between each step, the slide was  rinsed and dried with a hairdryer.  

This procedure determines whether the bacteria are Gram-positive or Gram-negative. Gram-positive bacteria appear blue or purple due to their cell wall being composed of 90% peptidoglycan, whereas Gram-negative bacteria appear pink or red due to their cell wall containing only 10% peptidoglycan and a high lipid content.

Result of Gram Staining

The result of gram staining can be seen on the image section.

Gram staining was done twice, once with the broth medium and another with the yogurt that was made.

Isolated culture

Shape: Cocci

Color: Purple

Yogurt from Stalks

Shape: Bacilli

Color: Purple

Conclusion

Isolation of a single bacterial colony was not achieved, as there are two different bacteria present.

Yogurt Making Procedure

Two types of starter cultures were prepared to compare the results of both methods: the original method proposed by the research, which utilized the chili stalks directly, and the newly developed method, which used isolated bacteria.

Analysis

Through the Gram staining method, the observation of purple staining on bacterial cells  signifies their Gram-positive nature. This staining pattern aligns with the characteristics  typically associated with lactic acid bacteria (LAB), particularly species like Lactobacillus,  which are known to be Gram-positive. Additionally, the rod-like shape of the observed  bacteria, further suggests the presence of LAB, as Lactobacillus species commonly exhibit  this shape. However, alongside the predominant presence of rod-shaped bacteria  resembling Lactobacillus, the identification of other shapes such as cocci, potentially  indicating the presence of Streptococcus, Lactococcus or related species, suggests  microbial diversity within the chili stalks. While Lactobacillus species are prevalent in dairy  fermentation, the presence of Lactococcus or Streptococcus indicates a broader microbial  community with the potential for other various activities or uses. Moreover, the ability of  these bacteria to reduce the pH of milk from its initial range of 6.4-6.8 to values ranging  from 5.23-5.46 for pure culture yogurt and 4.68-5.01 for stalk yogurt, although not in the pH  range of yogurt yet, demonstrates their fermentative ability. This pH reduction is  characteristic of lactic acid fermentation, wherein lactose in milk is converted into lactic acid  by LAB, resulting in the coagulation and acidification necessary for yogurt formation.  Furthermore, the determination of Total Titratable Acidity (TTA) provides insight into the  lactic acid content of the yogurt samples. The TTA for the yogurt made from pure culture  falling within the typical range of yogurt acidity (0.6-0.12%) confirms the presence of lactic acid-producing bacteria capable of acidifying milk. This finding supports the conclusion that  the bacteria present in chili stalks have the potential to serve as starter cultures for yogurt  production.  

In essence, the collective observations from Gram staining, pH reduction, and TTA  determination collectively suggest that the bacterial population inhabiting chili stalks harbors  organisms with the metabolic capabilities required for milk fermentation, demonstrating their  potential utility in yogurt production processes.

Conclusion and Future Work

• Gram staining revealed a diverse bacterial community within chili stalks, potentially harboring valuable resources for future exploration. 

• Although the optimal concentration for yogurt production from chili stalks remains undetermined, this study demonstrated the fermentative capacity of the present  Lactobacilli.  

• Comparison of methods indicated direct introduction of chili stalks into milk as more effective than the developed approach. Future research endeavors aim to refine methods for enhanced efficiency and safety surpassing the original proposed technique. 

• Further isolation of lactic acid bacteria from chili stalks can be done to discover utilization for other products.