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Production of probiotic cabbage juice by lactic acid bacteria

DOI:10.1016/j.biortech.2005.06.018
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Kyung Young Yoon at Yeungnam University
Kyung Young Yoon
Edward E Woodams
Edward E Woodams
Edward E Woodams
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Yong D Hang
Yong D Hang
Yong D Hang
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Abstract and Figures

Research was undertaken to determine the suitability of cabbage as a raw material for production of probiotic cabbage juice by lactic acid bacteria (Lactobacillus plantarum C3, Lactobacillus casei A4, and Lactobacillus delbrueckii D7). Cabbage juice was inoculated with a 24-h-old lactic culture and incubated at 30 degrees C. Changes in pH, acidity, sugar content, and viable cell counts during fermentation under controlled conditions were monitored. L. casei, L. delbrueckii, and L. plantarum grew well on cabbage juice and reached nearly 10x10(8) CFU/mL after 48 h of fermentation at 30 degrees C. L. casei, however, produced a smaller amount of titratable acidity expressed as lactic acid than L. delbrueckii or L. plantarum. After 4 weeks of cold storage at 4 degrees C, the viable cell counts of L. plantarum and L. delbrueckii were still 4.1x10(7) and 4.5x10(5) mL(-1), respectively. L. casei did not survive the low pH and high acidity conditions in fermented cabbage juice and lost cell viability completely after 2 weeks of cold storage at 4 degrees C. Fermented cabbage juice could serve as a healthy beverage for vegetarians and lactose-allergic consumers.
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Production of probiotic cabbage juice by lactic acid bacteria
Kyung Young Yoon, Edward E. Woodams, Yong D. Hang
*
Department of Food Science and Technology, Cornell University, Geneva, NY 14456, United States
Received 29 March 2004; received in revised form 14 June 2005; accepted 30 June 2005
Available online 24 August 2005
Abstract
Research was undertaken to determine the suitability of cabbage as a raw material for production of probiotic cabbage juice by
lactic acid bacteria (Lactobacillus plantarum C3,Lactobacillus casei A4, and Lactobacillus delbrueckii D7). Cabbage juice was inoc-
ulated with a 24-h-old lactic culture and incubated at 30 C. Changes in pH, acidity, sugar content, and viable cell counts during
fermentation under controlled conditions were monitored. L. casei,L. delbrueckii, and L. plantarum grew well on cabbage juice
and reached nearly 10 ·10
8
CFU/mL after 48 h of fermentation at 30 C. L. casei, however, produced a smaller amount of titratable
acidity expressed as lactic acid than L. delbrueckii or L. plantarum. After 4 weeks of cold storage at 4 C, the viable cell counts of L.
plantarum and L. delbrueckii were still 4.1 ·10
7
and 4.5 ·10
5
mL
1
, respectively. L. casei did not survive the low pH and high acid-
ity conditions in fermented cabbage juice and lost cell viability completely after 2 weeks of cold storage at 4 C. Fermented cabbage
juice could serve as a healthy beverage for vegetarians and lactose-allergic consumers.
2005 Elsevier Ltd. All rights reserved.
Keywords: Cabbage juice; Probiotic; Lactic acid bacteria; Lactobacillus casei;Lactobacillus delbrueckii;Lactobacillus plantarum
1. Introduction
Probiotics are defined as live microbial feed supple-
ment that beneficially affects the host by improving its
intestinal balance (Fuller, 1989). Most probiotic microor-
ganisms are lactic acid bacteria such as Lactobacillus
plantarum,Lactobacillus casei,Lactobacillus acidophilus,
and Streptococcus lactis (Sindhu and Khetarpaul, 2001).
Research has shown that addition of probiotics to food
provides several health benefits including reduction in
the level of serum cholesterol, improved gastrointestinal
function, enhanced immune system, and lower risk of co-
lon cancer (Berner and OÕDonnell, 1998; Rafter, 2003;
Saarela et al., 2002; McNaught and MacFie, 2001). Lac-
tic acid bacteria are commercially used as starter cultures
for the manufacture of dairy-based probiotic foods
(Heenan et al., 2002). Traditionally, probiotics have been
added to yogurt and other fermented dairy products, but
lactose intolerance and the cholesterol content are two
drawbacks related to their consumption. In recent years,
consumer demand for non-dairy-based probiotic prod-
ucts has increased, and probiotics have been incorpo-
rated into drinks as well as marketed as supplements in
the form of tablets, capsules, and freeze–dried prepara-
tions (Shah, 2001). Fruits and vegetables are rich in func-
tional food components such as minerals, vitamins,
dietary fibers, and antioxidants (phytochemicals). Fur-
thermore, fruits and vegetables do not contain any dairy
allergens that might prevent usage by certain segments of
the population (Luckow and Delahunty, 2004).
Cabbage is a cruciferous vegetable, which is rich in
minerals, vitamin C, dietary fibers, and especially phyto-
chemicals (Chu et al., 2002). The objective of this study
was to determine the suitability of cabbage as a raw
material for production of probiotic cabbage juice by
probiotic lactic acid bacteria.
0960-8524/$ - see front matter 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.biortech.2005.06.018
*
Corresponding author. Tel.: +1 315 787 2265; fax: +1 325 787
2284.
E-mail address: ydh1@cornell.edu (Y.D. Hang).
Bioresource Technology 97 (2006) 1427–1430
2. Methods
Cabbage (Brassica oleracea L. var. capitata L.) was
purchased from a local store and kept at 4 C prior to
use. Cabbage juice was obtained with a Loomis press
operated at 2000 psi and sterilized for 15 min at 121 C.
2.1. Probiotic lactic acid cultures
Lactobacillus casei A4,Lactobacillus debrueckii D7,
and Lactobacillus plantarum C3 were obtained from
the New York State Agricultural Experiment Station
Culture Collection, Geneva, New York. The cultures
were grown at 30 C for 24 h in MRS broth (dextrose
20.0 g/L; meat peptone 10.0 g/L; beef extract 10.0 g/L;
yeast extract 5.0 g/L; sodium acetate 5.0 g/L; disodium
phosphate 2.0 g/L; ammonium citrate 2.0 g/L; tween
80 1.0 g/L; magnesium sulfate 0.1 g/L, manganese sul-
fate 0.05 g/L).
2.2. Fermentation of probiotic cabbage juice
Fermentation experiments were conducted in test
tubes (25 ·200 mm), each containing 40 mL of sterile
cabbage juice. All samples were inoculated with a 24-h
culture (>10
5
CFU/mL) and incubated at 30 C for
72 h. Samples were taken at 0, 24, 48, and 72 h for chem-
ical and microbiological analyses.
2.3. Effect of cold storage on cell viability in probiotic
cabbage juice
After 72 h of fermentation at 30 C, the fermented
samples were stored at 4 C for 4 weeks. Samples were
taken at weekly intervals, and the viability of probiotic
cultures in probiotic cabbage juice was determined and
expressed as colony forming units (CFU/mL).
2.4. Chemical and microbiological analyses
The pH of probiotic cabbage juice was measured
with a pH meter. Total acidity, expressed as percent lac-
tic acid, was determined by titrating with 0.02 N NaOH
to pH 8.2. Sugar content was analyzed as glucose by the
phenol sulfuric acid method of Dubios et al. (1956). Via-
ble cell counts (CFU/mL) were determined by the stan-
dard plate method with Lactobacilli MRS medium after
48 h of incubation at 30 C.
2.5. Statistical analysis
All experiments were carried out in triplicate, and
each sample was analyzed in duplicate. The results are
expressed as mean ± S.D. (standard deviation). The
SAS statistical computer package was used to analyze
the experimental data (SAS Institute, Cary, NC,
USA). The values within rows that have no common
superscript are significantly different (p< 0.05) accord-
ing to DuncanÕs multiple range test (SAS Institute, Cary,
NC, USA). Any two means not marked by the same
superscript (for example, a and b or b and c within rows)
are significantly different (p< 0.05). Any two means
marked by the same superscript (for example, a and a
or b and b within rows) are not significantly different
(p< 0.05).
3. Results and discussion
All the three species of lactic acid bacteria, L. casei,
L. delbrueckii, and L. plantarum, were found capable
of growing well on sterilized cabbage juice without
nutrient supplementation. The time courses of lactic
acid fermentation of cabbage juice by L. casei,L. plan-
tarum,andL. delbrueckii are presented in Tables 1–3,
respectively. L. casei,L. plantarum and L. delbrueckii
grew rapidly on cabbage juice and reached nearly
10 ·10
8
CFU/mL after 48 h of fermentation at 30 C.
Extending the fermentation beyond 48 h did not result
in a significant increase in the viable cell counts of lactic
acid bacteria. Both L. plantarum and L. delbrueckii pro-
duced significantly more titratable acidity expressed as
lactic acid than L. casei. For example, L. plantarum
and L. delbrueckii produced nearly 1% titratable acidity
expressed as lactic acid after 72 h of fermentation
at 30 C. Under similar growth conditions, L. casei
Table 1
Time course of lactic fermentation of cabbage juice by Lactobacillus
casei
Time
(h)
pH Acidity
(% lactic acid)
Sugar
(mg/mL)
CFU/mL
0 5.0 ± 0.1
a
0.11 ± 0.01
d
45.6 ± 2.5
a
3.0 ± 0.2 ·10
6a
24 3.7 ± 0.0
b
0.38 ± 0.01
c
41.7 ± 1.4
b
6.3 ± 0.0 ·10
8b
48 3.4 ± 0.0
c
0.6 ± 0.03
b
39.5 ± 1.8
bc
12 ± 0.0 ·10
8c
72 3.4 ± 0.1
c
0.74 ± 0.02
a
36.5 ± 1.9
c
11 ± 0.1 ·10
8c
Means and standard deviations for n= 3. The experimental values
within rows that have no common superscript are significantly different
(p< 0.05) according to DuncanÕs multiple test range.
Table 2
Time course of lactic fermentation of cabbage juice by Lactobacillus
plantarum
Time
(h)
pH Acidity
(% lactic acid)
Sugar
(mg/mL)
CFU/mL
0 5.8 ± 0.0
a
0.12 ± 0.0
a
35.08 ± 0.09
a
8.0 ± 6.26 ·10
5a
24 4.8 ± 0.2
b
0.23 ± 0.06
b
37.10 ± 0.39
a
7.7 ± 3.41 ·10
8b
48 3.6 ± 0.0
c
0.76 ± 0.03
c
36.44 ± 3.77
a
15.3 ± 0.92 ·10
8c
72 3.6 ± 0.0
c
0.97 ± 0.03
d
19.33 ± 1.04
b
17.5 ± 7.05 ·10
8c
Means and standard deviations for n= 3. The experimental values
within rows that have no common superscript are significantly different
(p< 0.05) according to DuncanÕs multiple test range.
1428 K.Y. Yoon et al. / Bioresource Technology 97 (2006) 1427–1430
produced only 0.74% titratable acidity expressed as lac-
tic acid. It is probable that L. casei requires some essen-
tial growth nutrients which are deficient in cabbage juice
(Pederson and Albury, 1969). Earlier studies have
reported that an antibacterial substance is present in
cabbage (Pederson and Fisher, 1944; Dickerman
and Liberman, 1952; Kyung and Fleming, 1994a). The
growth inhibitory substance of fresh cabbage was sug-
gested to be carbohydrate in nature and of a low molec-
ular weight (Dickerman and Liberman, 1952). Kyung
and Fleming (1994b) reported that fresh juice of Cecile
cultivar cabbage was inhibitory to the growth of lactic
acid bacteria, and the inhibition was eliminated when
the cabbage was heated (steamed 10 min) before juice
extraction.
The data in Table 4 illustrate the effect of cold storage
on the viability of three species of lactic acid bacteria in
fermented cabbage juice. L. plantarum and L. delbrueckii
were capable of surviving in the fermented cabbage juice
at 4 C for several weeks. For example, the viable cell
counts of L. plantarum and L. delbrueckii were still
4.1 ·10
7
and 4.5 ·10
5
mL
1
, respectively, after 4 weeks
of storage at 4 C. However, L. casei was unable to sur-
vive the low pH and high acidity conditions in fer-
mented cabbage juice at 4 C and lost the cell viability
completely after only 2 weeks of cold storage. For the
maximum health benefits, the minimum number of pro-
biotic organisms in a food product should be 10
6
CFU/g
(Shah, 2001). Therefore, the viability of the lactic cul-
tures is the most important factor during refrigerated
or frozen storage. The viability of probiotic organisms
is dependent on the level of oxygen in products, oxygen
permeation of the package, fermentation time, and stor-
age temperature (Shah, 2000). The viability of probiotic
bacteria is also affected by inhibitory substances such
as lactic acid produced during production and cold
storage. Other factors for loss of viability of probiotic
organisms have been attributed to the decrease in pH
of the medium and accumulation of organic acid as a
result of growth and fermentation (Hood and Zottola,
1988; Shah and Jelen, 1990). In this study, we found
both L. plantarum and L. delbrueckii could survive the
high acidity and low pH in the fermented cabbage juice.
4. Conclusion
Three lactic acid bacteria, L. casei,L. plantarum,and
L. delbrueckii were examined for their ability to utilize
cabbage juice for cell synthesis and lactic acid production
without nutrient supplement. These lactic cultures grew
well in cabbage juice at 30 C, and the viable cell counts
reached nearly 10 ·10
8
CFU/mL after 48 h of fermenta-
tion at 30 C. Both L. plantarum and L. delbrueckii were
capable of surviving the low pH and high acidic condi-
tions in fermented cabbage juice during cold storage at
4C. In contrast, L. casei could not survive the low pH
and high acidity in fermented cabbage juice, and lost cell
viability completely after only 2 weeks of cold storage at
4C. From the results of this study, it is concluded that
L. plantarum and L. delbrueckii could be used as probi-
otic cultures for production of a healthy beverage from
cabbage for vegetarians or consumers who are allergic
to lactose present in probiotic dairy products.
Acknowledgements
Dr. Kyung Young Yoon received a post-doctoral fel-
lowship from Korea Science and Engineering Founda-
tion (KOSEF). This work was supported in part by
USDA Regional Project NE-1008.
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Maltodextrin esterified to laurate (ML) was synthesized enzymatically and compared to commercial sucrose laurate (SL) for microbial growth-inhibitory properties in food systems. There was broad microbial inhibition by ML in growth media, but practical information about various foods and storage temperatures is limited with no toxicological information. This research investigated the antimicrobial property of ML against two spoilage bacteria (Bacillus subtilis and Lactobacillus plantarum) in growth media and related food models stored at different temperatures (4 °C, 22 °C, and 35 °C). Additionally, in vitro cytotoxicity of ML was investigated in human colon adenocarcinoma HT-29 cells and human normal colon epithelial CCD 841 CoN cells using a standard colorimetric assay. ML was highly inhibitive against L. plantarum in apple juice at all temperatures tested. SL could inhibit both bacteria in the food models tested (milk, apple juice, tomato ketchup, French dressing) at 4 °C. ML was not observed to be cytotoxic to either cell line up to 500 μg/mL, whereas SL was evidently cytotoxic with IC50 ranging from 84 to 94 μg/mL. In conclusion, ML can be an alternative to commercial SL as a safe food additive in certain products.
... After the completion of the process, the juice had an optimal proportion of pigments, vitamins and minerals compared to the beetroot or carrot juice. Yoon et al. [57] fermented cabbage juice and concluded that Lactobacillus plantarum and L. delbrueckii were those lactic acid bacteria that should be used to carry out such a process. Traditional Korean kimchi is an interesting fermented product. ...
Fermented vegan foods
Article
  • Jan 2021
In recent years, an increase has been reported in consumer awareness of balanced diet and health prevention. This caused the consumer interest in functional foods to increase. The major functional foods are products that contain prebiotics and probiotics. The most often eaten probiotic product is classic yogurt, however the fermented dairy and non-dairy drinks tend to be more and more important. The increase in number and types of milk-free drinks on the market is due to increasing lactose intolerance among consumers. Additionally, in the developed countries gradually rises the number of people who are switching to veganism. The search for suitable substitutes for dairy milk and dairy products has become an important direction of scientific research and implementation projects in industry. The objective of the paper is to review the reference literature presenting results of research studies and experiments on the production and qualities of non-dairy probiotic products, that could be classified into vegan foods. New probiotic food in the form of drinks, which are already on the market or are still in research phase, are made of raw materials such as: vegetables, fruits, grains (oats, buckwheat, spelt wheat, corn, quinoa, amaranth), hazelnuts, coconuts, almonds, cashew nuts, and also sesame and hemp seeds. It is a big challenge for technologists to manufacture food products for vegans, because the vegan diet is more restrictive than a vegetarian one, therefore possibilities of using many raw materials are limited.
A Comprehensive Review on Microencapsulation of probiotics: Technology, Carriers and Current trends
Article
  • Dec 2022
Consumer interest in probiotics is significantly growing due to their positive impact on their health. Dairy products are common and most preferred probiotics "delivery vehicle" in the food industry. However, dairy products are associated with increased risk to people, with lactose intolerance, galactosemia, allergy to milk proteins, and high cholesterol levels. For such cases, non-dairy based probiotic foods could offer a good alternative. Among non-dairy foods, fruit juice is more dietary inclusive, convenient, and well accepted by all the age groups. Therefore, fruit juice could be used as a suitable non-dairy food carrier in probiotic delivery. Lactobacillus and Bifidobacterium are the two main strains used commercially worldwide for preparing probiotic products with proven health benefits. However, protecting the probiotic cells is the key for probiotic formulation in order to guarantee the survival. Therefore, various encapsulation techniques, cell viability, and suitable carrier materials in downstream processing and utilization are discussed in the review. Among different encapsulation techniques, spray drying emerged as an alternative technique for better utilization of probiotics in fruit juices with possibilities for industrial applications due to cost-effective and continuous process. Therefore, spray drying could be considered as an efficient encapsulation technique in food industry for fruit juice probiotification.
Pro-biotification of Underutilized Manila Tamarind Pulverized Seed Blended with Muskmelon
Article
Probiotics are live microorganisms that offer health benefits to consumers by improving their intestinal microflora balance. The word ‘probiotics’ is derived from the Greek term ‘pro’ and ‘biotic’ that means ‘for life’. Many dairy products and fruit pulp juices are added with probiotic flora. Many juices are also prepared using nutraceutically rich fruit seeds. Pitchellobium dulce seeds are rich in saponins, steroids, glycosides, lipids, phospholipids, polysaccharides, and glycolipids. It has anti-oxidant activity, anti-ulcer activity, free radical scavenging activity, anti-fungal activity, and anti-diabetic activity. In the present work, the probiotic juice was prepared using Pitchellobium dulce seed powder, muskmelon fruit pulp, and two Lactic acid bacteria (Lactococcus lactis and Lactobacillus acidophilus). The final product was evaluated for Colony Forming Unit (CFU), pH, sterility, taste, color, flavor, and texture. It was found that the shelf life of the juice at 40C is 3 months. It has shown that its direct consumption helps in reducing gastric ulcers and other gut-related problems, and if the extract is introduced into the nostril, it will reduce chest congestion. People who are allergic or intolerant to milk-based products can also drink the juice. This study aims to serve a delicious and nutritious drink to promote better health and nutrition for the population.
Processing of Probiotic Pomegranate Juice Using Lactic Acid Bacteria
Article
  • Sep 2022
The study was undertaken for production of probiotic pomegranate juice through its fermentation by two strains of lactic acid bacteria: Lactobacillus plantarum & L. acidophilus. Pasteurization of freshly prepared pomegranate juice at 80°Cfor 15 min was conducted to decrease the microbial population to below the detection limit. Fermentation was carried out at 37°C for 72 hrs. The parameters like, microbial population, pH, titrable acidity, and sugar (glucose) were measured during the fermentation period and viability of all the strains was also determined during the storage time at 4°C within week. The results indicated that L. plantarum & L. acidophilus decreased the pH sharply at initial stages of fermentation, sugar consumption was also higher and better microbial growth was also observed for these two strains during fermentation. L. acidophilus showed higher viability during the storage time than L. plantarum. Viable cells remained at their maximum level within 2 weeks but decreased dramatically after 3 weeks. The Pomegranate juice proved to be a suitable media for production of a probiotic drink.
A food-grade nanoemulsion for delivering probiotics and prebiotics
Chapter
  • Jan 2022
The positive impact of probiotics and prebiotics on human health has been demonstrated by various preclinical and clinical experiments. Therefore, they are used as supplements in a wide range of food products, including packaged foods, drinks, pickles, etc. However, preserving their beneficial effects in functional foods during food processing and storage is a major challenge in the food industrial sector. So, an effective delivery system is needed to acquire and retain the therapeutic properties and control the release of pro- and prebiotics. The nanoemulsion-based system is a revolutionary approach for sustaining the beneficial potential of pro- and prebiotics in functional foods and also for the effective delivery of these bioactives. This chapter discusses pro- and prebiotics together with the emerging role of food-grade nanoemulsions as an effective delivery system into functional food products toward improving their stability, specific target approach, and prolonged bioactivity.
Lactobacillus Use for Plant Fermentation: New Ways for Plant-Based Product Valorization
Chapter
  • Jul 2022
Today, plant production is increasing, but most industrial processes generate a lot of waste and by-products for which, in the current context, it is a priority to recycle or valorize them. One of the cheapest valorization routes is fermentation, in particular lactic fermentation by Lactobacillus species, which produces lactic acid and other molecules of industrial interest such as bioactive compounds such as anthocyanin, organic acid, peptides, or phenol, which are widely found in the plant matrix, mainly in cereals, grass, fruits, and vegetables. Bioactive compounds may exert beneficial health effects, such as antioxidant, anti-inflammatory, antimicrobial, or prebiotic activities. In addition, lactic acid fermentation can improve existing products and lead to new applications in food, livestock feeding and biotechnology, such as the production of lactic acid, protein, or silage. This chapter reviews the use of Lactobacillus strains in the fermentation process of many plant bioresources or by-products through their different bioactivities, active molecules, and applications.
Functional Foods and Health Claims Legislation: Applications to Dairy Foods
Article
The purpose of this paper is to consider the regulatory status of functional foods and claims that can be made regarding their health-promoting properties, with emphasis on the situation in the US. Specific examples highlight probiotics, prebiotics, and cultures-added dairy foods. In the US, there is no legal definition of a functional food. The US Food and Drug Administration regulates four categories of foods: conventional foods, foods for special dietary use, medical foods, and dietary supplements. Three types of health-related messages can appear under certain conditions on food labels. Nutrient content claims and approved health claims can appear on all qualifying foods, while structure/function claims are allowed on dietary supplements. (Medical foods are exempt from all nutrition labeling regulations.) Currently, there are no allowed health claims for probiotics, prebiotics, or cultures-added dairy foods in the US. Several approaches for taking advantage of ‘functional’ dairy foods in the US are outlined.
Probiotics in clinical practice: A critical review of the evidence
Article
The interaction of the gastrointestinal microflora with the human host has been the subject of considerable debate in the last decade. Manipulation of the enteric microflora with probiotic organisms has been attempted in a wide range of clinical settings, in the hope of achieving health benefits in the host. This review presents the evidence from human clinical trials of probiotics in the areas of diarrhoeal illness, inflammatory bowel disease, surgical prophylaxis, critical care and serum lipid modulation. With the exception of childhood viral diarrhoea, there is little evidence to support the use of probiotics in clinical practice at present. There are, however, sound theoretical reasons to support the role of probiotics in many other disease states.
Effect of Low pH on the Ability of Lactobacillus acidophilus to Survive and Adhere to Human Intestinal Cells
Article
Lactobacillus acidophilus was suspended in broth and buffer at pH 2, 3 and 4 and incubated at 37°C for 2 hr. In both broth and buffer pH 2, viable cell numbers decreased rapidly, and none were recovered after 45 min. At pH 4 in broth and buffer, the number of cells was not significantly reduced in 2 hr. In potassium phthalate buffer at p 3, no viable cells were recovered after 30 min, while in KCl buff and broth at pH 3, no significant reduction was seen. Cells subjected to low pH for up to 5 h were able to adhere to human intestinal cells in vitro. Exposure to low pH did not appear to disrupt the ruthenium red staining layer exterior to the cell wall.
Survival of Lactic Acid Bacteria and Their Lactases under Acidic Conditions
Article
Effects of sonication, survival, and β-galactosidase activity of four lactic cultures were investigated in pH 1.5-3.5 range. Lactobacillus delbruekii subsp. bulgaricus and Streptococcus thermophilus exhibited the highest β-galactosidase activity in skim milk and broth systems, respectively. The β-galactosidases from L. delbruekii subsp. bulgaricus, S. thermophilus, and L. acidophilus showed optimum activity in the neutral pH range and 55°C. Viable count of all four cultures decreased most rapidly at pH 1.5, but L. acidophilus and L. delbruekii subsp. bulgaricus survived better than the other organisms. The decrease of enzyme activity of unsonicated cultures with pH was slight, especially at pH 3.5. However, acidification of sonicated cultures to pH 3.5 or lower resulted in rapid and permanent loss of enzyme activity.