The Preservation of Lactobacilli by Freeze-drying

Abstract

SUMMARY : In developing a technique for the freeze-drying of lactobacilli particular attention was paid to the percentage survival rate; this was estimated by viable counts immediately after drying and after storage of the dried cultures. Themain factor influencing survival rate was the suspending medium, the best results being obtained in horse serum with 8 % added glucose. Forty-four cultures, fourteen freshly isolated, representative of a comprehensive collection of 452 lactobacilli (see Briggs, 1 9 5 3~) were freeze-dried in this vehicle using the apparatus and technique described. The majority of the survival rates were over 50 yo immediately after drying, and over 25 % after 6 months storage ; all but two of the freshly isolated strains dried well. Some of the dried cultures were tested again after a storage period of about 2 years and then showed satisfactory percentage survivals.For taxonomic studies of 452 strains of lactobacilli (Briggs, 1953a) cultures were maintained in yeast glucose litmus milk plus chalk at 4 O , which, although satisfactory for survival, involved considerable expenditure of media and labour; a less cumbersome method of maintenance was required, and the possibilities of freeze-drying were investigated.Since no outstanding method for lactobacilli had been described, the technique which had proved satisfactory for the freeze-drying of streptococci at this Institute was examined. The effects of different methods of preparing the culture, of various suspending media and of other modifications of the freezedrying technique were studied by determining the percentage survivals immediately after drying and at intervals during storage. A freeze-drying technique was evolved using as test organisms strains of lactobacilli particularly sensitive to drying. The method was then applied to forty-four cultures representative of the eight groups of Briggs (1953a), including fourteen freshly isolated strains, METHODSIn studying the conditions affecting the viability of lactobacilli during freezedrying and subsequent storage, the different factors involved were varied one by one. Deviations from, or comparisons with, the standard method described below are recorded in the appropriate places.Apparatus. The apparatus, illustrated in P1.

The preservation of lactobacilli: A comparison of three methods

“…The maintenance of cultures, particularly of Lactobacillus and related taxa, can be problematical. The most common method is to subculture in various media including soya bean medium (Cowan 1953) and milk media containing chalk (Sharpe & Fryon 1965;Bryan-Jones 1975), but viability cannot be retained for longer than 2-6 months. For long term preservation, lyophilization is popular especially for the maintenance of culture collections (Briggs et al 1955;Steele & Ross 1963).…”

“…The most common method is to subculture in various media including soya bean medium (Cowan 1953) and milk media containing chalk (Sharpe & Fryon 1965;Bryan-Jones 1975), but viability cannot be retained for longer than 2-6 months. For long term preservation, lyophilization is popular especially for the maintenance of culture collections (Briggs et al 1955;Steele & Ross 1963). Of these two methods, the former requires frequent subculturing and incurs a risk of genetic instability, contamination and loss of viability; the latter is an inconvenient method when frequent inocula are required since the contents of a complete ampoule must be resuscitated each time.…”

The Physiological and Serological Characters of Freeze-dried Lactobacilli

“…Immediately after freeze-drying (Briggs et al 1955) and 3-6 months later, twelve freeze-dried lactobacillus antisera were reconstituted and tested by precipitin ring tests against HC1 extracts of lactobacilli. Ten sera were found to have retained their potency and specificity; with one of these sera the potency after 6 months was markedly higher than that of the same serum stored in the liquid form, which had deteriorated.…”

“…Before freeze-drying a collection of strains of lactobacilli (Briggs, Tull, Newland & Briggs, 1955) it was therefore necessary to determine whether freeze-drying did in fact alter the physiological and serological chaTacters of the organisms dried. Elser, Thomas & Steffen (1935) freeze-dried strains of meningococci and gonococci and found them to be unaffected, and Cowan (1951and Cowan ( ,1954 found that cultures examined by biochemical tests before and after they were dried showed no significant differences.…”

Buttermilk Manufacture Using a Combination of Direct Acidification and Citrate Fermentation by Leuconostoc cremoris

“…Starter Culture Bacteria According to Briggs (1955) the suspending menstrum was the most important factor affecting the survival of bacterial cells during frozen storage. Gibson et al (1966) found that skim milk was the best suspending menstrum for lactic acid bacteria.…”