The pH of MRS culture medium (pH around 6.5) should be reduced to around 5 to 5.7 final pH. I prefer to use tartaric acid solution for this but whatever is normally used in your lab is fine. If pH is 5.0, you may not get growth of enterococci. I'd probably go for pH 5.2. Since your product is acidic, you will have to experiment and determine how much your product brings down the pH of the culture media so you can adjust the pH accordingly. (As pH of agar is decreased, you can get softening of the agar due to hydrolysis of polysaccharide chains in agar). You may have to play around with a pH that works best for you. Check the effect of addition of sorbic acid on final medium pH (it may not have an effect but I would check). Sorbic acid is MORE effective as pH decreases since you want it in the un-dissociated state at or below it's PKA for optimal activity. You already have sodium acetate in MRS which also acts as an antimicrobial for gram negatives and yeasts as the pH is reduced. Adjust pH of medium so you get the final pH you want. The 1:10 dilution of your high acid product may be the only one that really causes a problem with pH.. Higher dilutions shouldn't affect pH of medium that much. But, always check at the beginning of an experiment and record it for future reference. Check before and after autoclaving the first few times to be sure.

You may want to consider M-17 culture medium for isolation of lactics if you continue to have problems. The big difference is that M-17 does not have simple carbohydrates (glucose or dextrose, lactose, etc.). The 20% dextrose in MRS can be a real problem since almost all bacteria and yeast can metabolize it.

M-17 is rich in nutrients but doesn't contain simple sugars. When I use M-17 agar, all the above apply. I often add 10 to 15 g/L of mannitol to M-17 since many lactics metabolize it happily but few gram negatives can metabolize it quickly or at all. Yeasts can metabolize a number of sugars but the complex ones will slow them down. Most Yeasts really prefer simple sugars like dextrose.

The key to effective isolation of lactics is to design a system that gives them a boost over competitors at the beginning. Once they start growing and get into log phase, the competition doesn't really have much of a chance. A buffering system can really help if you're getting poor recovery since some lactics can quickly bring the pH down through their metabolites and inhibit the growth of other lactics. A simple acetate buffering system (pH 3.6 to 5.6) at the desired pH can be helpful. This can also help to prevent problems with high acid product. .Phosphate buffers don't really work that well below pH 5.7.

Hope you find this helpful.

I forgot to mention that agar culture media should be acidified (pH 5 to 6 range) AFTER autoclaving immediately prior to pouring plates. This helps reduce the softening of the agar that I mentioned in my previous response to your question. The hydrolysis of agar at lower pH is accelerated by heat. The softening can be a real pain since its very hard to spread plate and the medium can actually form a semi-solid rather than solid product.

Thank you so much for your assistance. I am going to take some samples of my product at the end of this month and will start troubleshooting using what you have said. I will give you feedback.

Same problem is being faced in my laboratory even during maintenance of pure culture isolates. I have also found that the pH adjustment gives positive results but not satisfactory. However, I have not tried sorbic acid and tartaric acid so far.

To prevent growth of yeast you can add 0,01% cyclohexamide to MRS medium